Catering to all applications, skill sets

Varioskan LUX is a versatile tool for busy labs. Configure the instrument to your needs, then upgrade when your research focus changes. Supports the following measurement technologies:

  • Absorbance (UV-Vis, including pathlength correction)
  • Fluorescence Intensity (including FRET)
  • Luminescence (direct and filtered, including BRET)
  • AlphaScreen / AlphaLISA
  • Time-resolved fluorescence (including TR-FRET, hTRF)

Flexible wavelength selection

The instrument selects the measurement wavelength using filters or monochromators, depending on which is optimal for each measurement technology.

  • Monochromators in absorbance and fluorescence intensity
  • Filters in AlphaScreen and time-resolved fluorescence
  • Luminescence without wavelength selection (filters can be used if required)

The instrument also allows spectral scanning for ultimate flexibility for identifying the optimal measurement wavelength for any assay, now and in the future.

Automatic dynamic range selection

Many multimode readers require the user to adjust PMT gain voltages manually in order to achieve the optimal sample signals for fluorescence and luminescence assays. But this manual trial and error process takes time, and can result in loss of sensitivity or over-saturation of signals. The Varioskan LUX multimode reader has an automatic dynamic range selection feature designed to automatically select the optimal reading range based on the signal intensity in each well - easing assay setup and helping you get optimal results, every time.

   Read our dynamic range selection smart note

Figure 1. Example of the reached dynamic range and sensitivity in a fluorometric measurement of fluorescein concentration series, performed by Varioskan LUX and a microplate reader from another manufacturer.

Smart safety controls

The potential for experimental errors—from user mistakes to power outages—are a frustrating reality of microplate reading. But the Varioskan LUX instrument is designed with automatic safety checks designed to alert you of potential problems before they happen.

 Read our smart safety controls smart note

Hood-lifted front view of the two reagent dispensers inside the Varioskan LUX multimode reader
The convenient automatic dispensing capability offered by the Varioskan LUX multimode reader makes it important to sense whether the right plate is present.

Integrated gas control

Graph of experimental kinetic data with and without carbon dioxide to illustrate integrated gas control capabilities of the Varioskan LUX Multimode Reader

Some cell-based assays require precise control over carbon dioxide and oxygen concentrations to keep the atmosphere optimal for cell growth. The ability to control O2 and CO2 levels in your microplate reader replaces the need for constant handling of plates between the incubator and reader, and helps to ensure that cells remain alive throughout the experiment.

  Read our integrated gas control smart note

Figure 2. Kinetic study of the growth of HeLa cells was performed in the Varioskan LUX instrument for 72 hours with and without internal CO2 level control. Average curves of baseline-subtracted fluorescence values of 48 replicates are plotted.

Evaluation of the toxic effects of PFOS in hESCs differentiating into cardiomyocytes

In this application note, R. Yang, et.al. studied the effects of an environmental toxin, PFOS, on the differentiation of hESCs into cardiomyocytes as a model for understanding the impact of PFOS exposure on heart development during pregnancy.

We demonstrate the benefits of using the Varioskan LUX Multimode Microplate Reader and the EVOS FL Imaging System to detect changes in cell viability and mitochondrial membrane potential (MMP) of differentiating hESCs upon treatment with PFOS.

Cell viability on differentiation days 2, 4, and 8 after chemical treatment was measured with Invitrogen PrestoBlue HS Cell Viability Reagent, a one-step resazurin-based cell viability assay, which is a cost-effective, sensitive, and simple method to detect cellular viability. PFOS has been shown to cause mitochondrial toxicity and depolarization of the mitochondrial membrane. Thus, we measured MMP by staining the differentiating cardiac cells with JC-1 dye (part of the Invitrogen MitoProbe JC-1 Assay Kit) after PFOS treatment.

Schematic diagram of experimental approach for evaluating potential PFOS toxicity, showing the steps from cell culture to treatment to cell imaging
Figure 3. Experimental layout for evaluation of potential developmental cardiac toxicity of PFOS. hESCs were seeded as single cells in 96-well plates and differentiated into beating cardiomyocytes for 8 days, as indicated. Cell viability and MMP were detected with the Varioskan LUX Multimode Microplate Reader and EVOS FL Imaging System. In the schematic of the 96-well plate, blue wells are filled with DPBS (the wells from B10 to G10 were filled with CDM3 medium only prior to cell viability measurements); white wells are filled with cells treated with DMSO; orange wells are filled with cells treated with different concentrations of PFOS.

FluoroSpot Pre-Screening with Thermo Scientific Microplate Readers

Thumbnail of page 1 of web-based app note: FluoroSpot Pre-Screening with Thermo Scientific Microplate Readers

In this technical note, we introduce a new image-independent procedure for the initial evaluation of FluoroSpot plates, which can be performed with Thermo Scientific fluorescence microplate readers (Varioskan LUX Multimode Microplate Reader, Fluoroskan Microplate Fluorometer or Fluoroskan FL Microplate Fluorometer and Luminometer). For the current study, FluoroSpot assays were evaluated using the Varioskan LUX multimode reader. The procedure involves a rapid measurement of the fluorescence intensity within a defined arrangement of multiple points on the bottom of every well. The limit of detection (LOD) of this FluoroSpot pre-screening is calculated here for two model cytokines (IFN-γ and IL-2). The limitations and applicability of this image-independent FluoroSpot pre-screening method are also further discussed.

ELISpot and FluoroSpot assay workflow represented as simplified five-step graphic concluding with both brightfield (ELISpot) and fluorescence (FluoroSpot) images of results
Figure 4. The ELISpot/FluoroSpot assay workflow.

Monitoring cell health with alamarBlue and PrestoBlue reagents using the Varioskan LUX Multimode Microplate Reader

In this application note, the benefits of pairing alamarBlue and PrestoBlue reagents with the Varioskan LUX Multimode Microplate Reader are demonstrated. Both viability assays are simple to perform, and the generated signal (fluorescence or absorbance) typically can be read in a conventional microplate reader. However, utilizing the Varioskan LUX Multimode Microplate Reader and SkanIt Software significantly enhances the usability of these resazurin-based workflows (Figure 1), allowing fast readout of signals and immediate access to powerful data processing steps such as curve fitting and useful calculations, including cell viability percentages and cytotoxic potency.

Black microplate with clear-bottom wells treated with alamarBlue or PrestoBlue reagent exhibiting a rainbow-style coloring going left to right green-blue-purple
Screenshot in SkanIt Software of the heatmap data visualization of the same microplate from (A), read using fluorescence mode of the Varioskan LUX multimode reader

Figure 5. Cell health monitoring with alamarBlue and PrestoBluereagents. (A) Plate and (B) SkanIt Software data visualization.

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The optimized solution to quantify and visualize cells

See how the Thermo Scientific Varioskan LUX multimode microplate reader and the Invitrogen EVOS Cell Imaging System work together to provide simplified versatility and stunning imagery.

Take a Closer Look

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Next Generation of Multimode Microplate Reading

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Absorbance
Plate types 6, 384 well plates
Wavelength selection Double monochromators
Wavelength range 200–1,000 nm
Light source Xenon flash lamp
Read out range 0–6 Abs
Linear measurement range 0–4 Abs (96-well plate) at 450 nm, ± 2%
0–3 Abs (384-well plate) at 450 nm, ± 2%
Accuracy 0.003 Abs or ± 2%, at 200–399 nm (0–2 Abs)
0.003 Abs or ± 1%, at 400–1,000 nm (0–3 Abs)
Precision SD <0.001 Abs or CV <0.5%, at 450 nm (0–3 Abs)
Fluorescence Intensity
Plate types 6–1,536 well plates
Wavelength selection  Double excitation and emission monochromators
Excitation wavelength range 200–1,000 nm
Emission wavelength range  270–840 nm
Light source Xenon flash lamp
Sensitivity Top reading: <0.4 fmol fluorescein/well (black 384-well plate)
Bottom reading: <4 fmol fluorescein/well, (clear bottom black 384 square well plate)
Dynamic range Top reading >6 decades
Bottom reading >5.5 decades
Time-resolved fluorescence
Plate types 6–1,536 well plates
Wavelength selection Filters (spectral scanning with double excitation and emission monochromators)
Excitation wavelength range Fixed to 334 nm (spectral scanning 200–840 nm)
Emission wavelength range 400–700 nm (spectral scanning 270–840 nm)
Light source  Xenon flash lamp
Sensitivity <1 amol Eu/well (white low volume 384 well plate)
Dynamic range >6 decades
Luminescence
Plate types 6–1,536 well plates (spectral scanning 6, 384 well plates)
Wavelength selection Direct or filters (spectral scanning with double monochromators)
Wavelength range  360–670 nm
Sensitivity <7 amol ATP/well (white 384-well plate)
Dynamic range >7 decades
AlphaScreen
Plate types 6, 1,536 well plates
Wavelength selection  Filters
Excitation wavelength range Fixed to 680 nm
Emission wavelength range 400–660 nm
Light source  LED
Sensitivity <100 amol phosphotyrosine/well (white 384-well plate)
Dispensing
Plate types 6, 384 well plates
No. of dispensers None, one or two
Syringe size  1 ml (standard), 5 ml (optional)
Dispensing volume 2–5,000 μl, in 1 µl increments (1 ml syringe)
5–25,000 μl, in 5 µl increments (5 ml syringe)
Accuracy <1 μl with 50 μl (0.4 mm tip)
<0.2 μl with 5 μl (0.25 mm tip)
Precision <1 μl with 50 μl (0.4 mm tip)
<0.25 μl with 5 μl (0.25 mm tip)
Dead volume Reagent loss <100 μl, Total tubing volume <800 μl
Incubator and Shaker
Temperature range From ambient + 4°C to 45 °C
Shaking type  Orbital
Integrated Gas Module
CO2 concentration range 0.1–15%
CO2 concentration stability  ± 0.3% at 5% CO2
O2 concentration range 1–21 %
O2 concentration stability  ±0.3% at 1% O2
General Features
Measurement modes End-point, kinetic, spectra, multipoint and kinetic spectra
Measurement speed Reads a 96-well plate in 15 s, a 384-well plate in 45 s, and a 1,536-well plate in 135 s (minimum times)
Interface  PC software (SkanIt software)
Dimensions (D x W x H) 58 x 53 x 51 cm (23 x 21 x 20 in)
Weight  54–59 kg (119–130 lb), depending on configuration

Low-volume quantification of nucleic acids and proteins with Thermo Scientific µDrop and µDrop Duo Plates

Introduction

Photometry in the UV range is a common way to quantify nucleic acids and proteins in a sample. Both nucleic acids and proteins absorb UV light. In the case of nucleic acids, the nitrogenous bases present in all nucleotides have a clearly distinguishable absorption maximum at 260 nm. On the other hand, in the case of proteins, the light absorptive properties at 280 nm are limited to few amino acids, specifically the aromatic amino acids tryptophan (Trp) and tyrosine (Tyr), and to a lesser extent cysteine groups forming disulfide bonds (Cys-Cys). Therefore, the absorption of proteins and peptides at 280 nm is proportional to the content of these amino acids.

These UV-absorptive properties make it possible to use direct photometry to quantify the concentrations of nucleic acids and proteins. Such calculations are performed using Beer-Lambert’s Law, which describes that the absorbance of a certain nucleic acid or a protein depends on the molecule’s concentration, the molecule’s absorptivity coefficient (given by the intrinsic absorptivity properties of such molecule) and the pathlength of the incident light. The use of this equation specifically for calculation of nucleic acids and proteins is shown on Table 1.

Table 1. Calculation of nucleic acids and protein concentrations using direct absorbance measurements, according to Beer-Lambert’s Law.

  Nucleic acids Proteins
Example case Double stranded DNA (dsDNA) Bovine serum albumin (BSA)
Equation for the calculations

Where:
C = concentration of dsDNA (µg/mL)
A260 = absorbance value at 260 nm of sample and blank
A320 = absorbance value at 320 nm of sample and blank
L = light pathlength (cm)
εdsDNA = extinction coefficient of dsDNA = 0.02 (µg/mL)-1cm-1
Note: Ratio of is 50 (µg/mL) cm

Where:
C = concentration of BSA (mg/mL)
A280 = absorbance value at 260 nm of sample and blank
A320 = absorbance value at 320 nm of sample and blank
L = light pathlength (cm)
ε1% = mass extinction coefficient of BSA = 6.7 (mg/mL)-1cm-1 for 1% (10 mg/mL) BSA solution

Equation for molar extinction coefficient for ε proteins   Molar extinction coefficient ε for any protein can be easily calculated using following equation:
ε=(nW * 5500) + (nY * 1490) + (nC * 125)
Where:
W: tryptophan, Y: tyrosine, C: cysteine
n: number of each residue present in the protein
(5500, 1490, and 125: molar absorptivity at 280 nm of W, Y, and C, respectively)

The calculations of nucleic acids and proteins are performed based upon absorbance measurements at 260 nm and 280 nm, respectively, but an additional measurement at 320 nm is typically carried out with the purpose of background subtraction. Measuring at 320 nm allows excluding impurities in the sample, for example arising from the presence of magnetic beads in the sample.
 

Low-volume quantification with Thermo Scientific µDrop Plate and µDrop Duo Plates

The amount of sample available for nucleic acid and protein analysis is often low. To address that challenge, Thermo Scientific offers the µDrop and µDrop Duo Plates, which enable direct photometric measurements at a microliter scale (Figure 1). The Drop and µDrop Duo Plates consist of two separate measurement locations: the left side is intended for measurements of low-sample volumes and the right side is for measuring cuvettes. The cuvette slot is used to perform photometric measurements (including kinetic studies) with standard cuvettes that are covered with stopper and placed in a horizontal position.

The low-volume measurement area consists of two or four quartz slides: one or two pairs of the top clear quartz slide and the bottom quartz slide, which is partially Teflon-coated. The bottom slide contains a matrix array position where samples are pipetted. In the case of the µDrop Plate, there are 16 samples positions arranged in a 2 x 8 matrix, while in the case of the µDrop Duo Plate, there are 32 sample positions arranged in 2 separated arrays also in a 2 x 8 matrix (Figure 1). The pathlength of the µDrop Plate is fixed, and the nominal value is 0.05 cm. However, the pathlength can vary between 0.048 and 0.052 cm, and verified pathlength for each μDrop Plate is indicated on the quality control measurement report delivered with the μDrop Plate. Same applies to the µDrop Duo Plate (with 32 samples), but in this case, there are two separate pathlength values, one for each 2 x 8 array. These exact pathlength values should always be used for the concentration calculations, instead of the nominal value of 0.05 cm, in order to obtain more accurate results.

top view of the plates showing the quartz slides and the positions for pipetted samples

Figure 1. μDrop Plate and the μDrop Duo Plate.

Compared to a normal cuvette having a pathlength of 1 cm, the pathlength of the μDrop Plate or μDrop Duo Plate is 20 times shorter (0.05 cm). Because of this, there is roughly a 20 times difference between the concentrations that can be measured with a typical cuvette versus the μDrop and μDrop Duo Plate. This means that the detection limit of any given analyte is about 20 times higher in the μDrop and μDrop Duo Plate than with cuvettes, when measured in the same instrument. On the other hand, a shorter pathlength allows for lower sample volumes to be measured. In the case of the μDrop and μDrop Duo Plate, a sample volume down to 2 µL can be used. It is possible to measure nucleic acid and protein concentrations from a few to thousands of micrograms per microliter using these devices together with a photometer that has sufficiently high precision and a wide linear range, such as the Thermo Scientific Multiskan SkyHigh Microplate Spectrophotometer. Finally, any photometric measurement device always has a certain background absorption. Therefore, blank subtraction is necessary when photometric quantification of the sample concentrations is performed.


Materials and methods

To calculate the detection limits for nucleic acid quantification (dsDNA), 11 dilutions were made of a DNA stock solution of Herring sperm DNA (Promega, D1816) using TE-buffer, (Invitrogen 10 mM Tris-HCl, 0.1 mM EDTA), to cover concentration range between 50 and 3500 µg/mL. To calculate the detection limits for protein quantification (BSA), 9 dilutions were made of a BSA stock solution (Sigma, A7030) to distilled water to cover concentration range between 0.5 and 100 mg/ml. The blanks as well as dsDNA or BSA samples were measured by pipetting 4 µL into the measurement areas of the μDrop Plate and the µDrop Duo Plate. In case of both μDrop and µDrop duo plates, 2 blanks were used. All measurements were performed using Multiskan SkyHigh Microplate Spectrophotometer with touchscreen and cuvette.

As indicated earlier, to ensure that proper concentration calculations, the exact pathlength values of the μDrop Plate or µDrop Duo Plate need to be provided. These values were defined in Multiskan SkyHigh settings by selecting “Settings” from the Home screen and then selecting “μDrop Plates” as indicated in Figure 2. The final concentrations reported by the instrument will be calculated with these pathlength values, and the user will not need to perform any correction steps, afterwards. Ready-made assay protocols in the Multiskan SkyHigh user interface for dsDNA or BSA quantification were used to perform the assays.

User interface screenshot showing how users specify pathlengths into the software

Figure 2. Setting-up the pathlength values of the μDrop Plate and μDrop Duo Plate in the User Interface of the Multiskan SkyHigh Microplate Spectrophotometer.

When the Multiskan SkyHigh model without touch screen is used, the dsDNA concentration calculations need to be done using the Thermo Scientific SkanIt software, which controls all the Thermo Scientific microplate readers. For that purpose, in “Plate Layout”, the appropriate plate format should be selected from the dropdown menu (μDrop Plate or μDrop Duo Plate). In the case of the μDrop Duo Plate, it is possible to add two values as the pathlengths on the two separate measurement areas may differ. See an example of the performed calculations in Figure 3.

Screenshot of software showing the results of the dsDNA concentration calculations

Figure 3. Setting-up the pathlength values of the μDrop Duo Plate in the SkanIt software for calculations of the dsDNA concentrations using Multiskan SkyHigh Microplate Spectrophotometer. If the calculation is performed without 320 nm subtraction, the factor 50 is simply added to the pathlength correction step of the 260 nm measurement data.

Multiskan SkyHigh models with UI, the ready-made sessions have all the necessary calculations. The results automatically contain the concentration, purity ratios and the sample spectrum, Figure 4 below.

Calculations results screen showing the dsDNA measurements and corresponding concentration and purity values

Figure 4. An example data set of a µDrop duo plate dsDNA measurement.


Results and discussion

Detection range of the µDrop Plate and µDrop Duo Plates

The detection range is given by the lowest and highest possible concentrations that can be reliably measured with a given instrument. Therefore, these two extremes of the detection range are instrument dependent and they can be theoretically calculated from the instrument’s performance specifications. In this note, however, we focus on the detection ranges of nucleic acids and proteins, as experimentally measured using IUPAC recommendations. Often the limiting factor is the lowest end of the detection range, since unknown samples that have too low concentrations of nucleic acids or proteins (falling under the lowest part of the detection range) cannot be simply accurately estimated using photometric detection with the µDrop Plate or µDrop Duo Plates. Often, because most microplate spectrophotometers in the market have very similar detection ranges, the only solution in such cases is to change the detection technology and use instead fluorescence-based detection, which is known to be considerably more sensitive. By contrast, for samples with too high concentration values (falling outside of the detection range), a simple solution is to dilute the samples, so that they can fall within the detection range, and thus can be accurately measured.

Limits of Detection (LOD) and Limits of Quantification (LOQ) for measuring nucleic acids and proteins

The lowest part of the detection range curve indicates the assay sensitivity, which can be assessed, according to the IUPAC, with two different parameters: Limit of Detection (LOD) and Limit of Quantification (LOQ). The LOD is defined as the lowest quantity or concentration of analyte that can be separated from the background or blank values. The LOD value means that the presence of the analyte can be detected with statistical significance, but the analyte cannot be quantified as an exact value. On the other hand, the LOQ is defined as the lowest quantity or concentration of the analyte at which quantification is possible with statistical relevance. In practice, LOD is the limiting value in qualitative assays where the question “is there any analyte in my sample or not?” needs to be answered. In such cases, LOQ is the limiting value that the user can measure as the concentration of the analyte in quantitative assays. Both LOD and LOQ calculations assume that both blank samples and replicates of the analyte are normally distributed. Signals at the LOD are defined as the blank mean + 3 * standard deviation of the blank, while signals at the LOQ are defined as blank mean + 10 * standard deviation of the blanks. Experimental calculations of the LOD and LOQ values is detailed in Table 2.

Table 2. Mathematical definitions of Limits of Detection (LOD) and Limits of Quantification (LOQ).

  LOD LOQ
Equations for the calculations

 

Where:
k = 3
SD blanks = standard deviations of instrument readings taken on assay blanks
m = slope of a graph of instrument’s blanked signal vs. concentration of the analyte on the linear range, as calculated by linear regression

 

Where:
k = 10
SD blanks = standard deviations of instrument readings taken on assay blanks
m = slope of a graph of instrument’s blanked signal vs. concentration of the analyte on the linear range, as calculated by linear regression

The calculated values for LOD and LOQ for both nucleic acids and proteins, exemplified with dsDNA and BSA are reported in Table 3.

Table 3. Limits of Detection (LOD) and Limits of Quantification (LOQ) calculated for nucleic acids (dsDNA) and proteins (BSA) using the µDrop Plate or µDrop Duo Plates.

    µDrop Plate µDrop Duo Plate
Nucleic acid (dsDNA) LOD 2.0 µg/mL* 2.9 µg/mL*
LOQ 6.5 µg/mL * 9.8 µg/mL *
Protein (BSA) LOD 0.4 mg/ml 0.4 mg/ml
LOQ 1.5 mg/ml 1.5 mg/ml

*This concentration difference between µDrop and µDrop Duo plate is statistically meaningless because it is caused by random approx. 0.001 Abs difference in the measured Abs 260 nm value. This A260 nm difference is below the precision specification of the instrument and therefore the difference in LOD and LOQ values between these two µDrop plate types is individual random happening in this test.


Linearity ranges

The dsDNA experiment described above is also used as an example to demonstrate the linear range of the Multiskan SkyHigh and µDrop plate system in practice.

As mentioned in the detection range section, with a measurement device with fixed pathlength, like the µDrop plates, the maximum measurement range is always determined by the instrument. The lower part is determined by the precision of the blank (LOD) and the upper part by the linear range of the instrument. So, for Multiskan SkyHigh, which is specified to be linear up to 2.5 Abs, the theoretical maximum concentration is 2.5 x 50 μg/ml / 0.050= 2500 μg/ml

The absorbance values of both µDrop and µDrop Duo plates are plotted as function of the theoretical concentration of the dsDNA samples in Figure 5.

Graph of absorbance vs concentration for µDrop and µDrop Duo plates compared to theoretical concentrations of sample

Figure 5. µDrop and µDrop Duo plate absorbance A260 nm values as function of the theoretical dsDNA concentration.

This data shows that the linear range up to 2.5 Abs well applies also to Multiskan SkyHigh with the µDrop plates in dsDNA A260 nm measurements.


Conclusions

Multiskan SkyHigh combined with μDrop or μDrop Duo Plate is an ideal tool for quick and easy concentration measurements of DNA, RNA, protein samples, or spectral scanning with very low sample consumption. Multiskan SkyHigh, µDrop plates and ready-made sessions for nucleic acid and protein analysis in the UI and in the SkanIt cloud library offer a very easy to use approach to these measurements. Samples are easy to pipette onto the μDrop Plates with a single or an 8-channel pipette. The plates are easily wiped clean, making it convenient to be used in serial measurements.

Read about the µDrop and µDrop Duo plates

Q: What instrument features can I add later as an upgrade?

A: Absorbance and fluorescence intensity are included with every instrument. The measurement technologies of the LAT module (Luminescence, AlphaScreen and Time-Resolved Fluorescence) can be upgraded. The only limitation is that the LAT module cannot exist without luminescence technology. Thus, if the researcher wants AlphaScreen and/or time-resolved fluorescence, they will also get luminescence. The dispensers and integrated gas module are also upgradable. Bottom reading, however, must be included in the original order, and cannot be added later.

Q: For what measurement technologies do I need to purchase and install filters?

A: In time-resolved fluorescence and AlphaScreen the instrument requires filters for wavelength selection. The excitation filters for both technologies are built-in, but the emission filters must be ordered and installed separately. When you install filters to the LAT module, you have to enter the filter information to the instrument via SkanIt software. Absorbance and fluorescence intensity technologies use monochromators for wavelength selection, thus filters are not necessary. Most of the luminescence assays do not require any wavelength selection. But if required, filters can be installed.

Q: What measurement technologies allow spectral scanning?

A: The instrument includes monochromators to allow spectral scanning in all measurement technologies except AlphaScreen. The instrument must have the corresponding measurement technology onboard to be able to run spectral scanning.

Q: How do I choose the right plate adapter?

A: The plate adapter lifts the microplate to optimal height for measurement and dispensing. Choose the adapter based on the plate format (96-well, 384-well, etc.) and whether or not the plates have lids. When starting a measurement, the SkanIt software checks that the installed plate adapter is compatible with the plate template selected, and informs you if there’s a mismatch.

Q: When should I use bottom reading?

A: Bottom reading should be used when measuring adherent cells that are at the bottom of the well. From the bottom side the excitation light can be focused more accurately on the cell layer. Bottom reading is available only in fluorescence intensity measurements with an instrument that has the bottom reading option.

Q: There are two dispensing positions in the instrument: F and L. How do I choose the right one?

A: Either position can be used with either dispenser. If the assay reaction is fast and the signal changes quickly after triggering the reaction by dispensing, select the dispensing position which points at the measurement position of the technology you will measure. If you use a dispensing position that does not point at the correct measurement position, the instrument makes an extra plate movement between dispensing and measurement. See Varioskan LUX user manual for more information on which measurement technologies match with dispensing position F or position L.

Q: Varioskan LUX is able to dispense and measure simultaneously. In what type of assay is this important?

A: It is important in fast kinetic assays where the peak signal is reached soon after reaction triggering. If the peak signal is reached in less than a second after dispensing, for example, simultaneous dispensing and measurement allows you to follow the signal progress from the very start of the reaction without losing the signal peak.

Q: How does Varioskan LUX incubator prevent condensation from forming on the microplate lid, and why is it important?

A: Condensation on the microplate lid affects measurement results. In the Varioskan LUX, the microplate is surrounded by temperature-controlled heaters. The upper element is slightly warmer than the lower element to avoid condensation on the plate lid.

Q: Can I install SkanIt software on several computers?

A: Yes, there is no limit on software licenses within the same customer organization. Measurement session files can even be exported and imported between SkanIt software installed on different computers, allowing researchers to work with the software outside the laboratory.

Q: How does the Drug Discovery Edition of SkanIt software differ from the Research Edition?

A: The Drug Discovery Edition offers features required for compliancy with FDA 21 CFR Part 11, a must for the pharma industry. Those special software features provide an audit trail, require an electronic signature, and a prompt to provide a reason for any changes made. Apart from these three special features, the Research Edition and the Drug Discovery Edition have the same features and functionalities.

Q: If I abort the run before it’s finished, does the measurement data disappear?

A: No, the data up to the time of abortion is saved. SkanIt software saves the measurement data automatically and constantly to the software database during a run. If a long kinetic measurement is aborted either unexpectedly or intentionally, the data does not disappear.

Automatic dynamic range selection

Many multimode readers require the user to adjust PMT gain voltages manually in order to achieve the optimal sample signals for fluorescence and luminescence assays. But this manual trial and error process takes time, and can result in loss of sensitivity or over-saturation of signals. The Varioskan LUX multimode reader has an automatic dynamic range selection feature designed to automatically select the optimal reading range based on the signal intensity in each well - easing assay setup and helping you get optimal results, every time.

   Read our dynamic range selection smart note

Figure 1. Example of the reached dynamic range and sensitivity in a fluorometric measurement of fluorescein concentration series, performed by Varioskan LUX and a microplate reader from another manufacturer.

Smart safety controls

The potential for experimental errors—from user mistakes to power outages—are a frustrating reality of microplate reading. But the Varioskan LUX instrument is designed with automatic safety checks designed to alert you of potential problems before they happen.

 Read our smart safety controls smart note

Hood-lifted front view of the two reagent dispensers inside the Varioskan LUX multimode reader
The convenient automatic dispensing capability offered by the Varioskan LUX multimode reader makes it important to sense whether the right plate is present.

Integrated gas control

Graph of experimental kinetic data with and without carbon dioxide to illustrate integrated gas control capabilities of the Varioskan LUX Multimode Reader

Some cell-based assays require precise control over carbon dioxide and oxygen concentrations to keep the atmosphere optimal for cell growth. The ability to control O2 and CO2 levels in your microplate reader replaces the need for constant handling of plates between the incubator and reader, and helps to ensure that cells remain alive throughout the experiment.

  Read our integrated gas control smart note

Figure 2. Kinetic study of the growth of HeLa cells was performed in the Varioskan LUX instrument for 72 hours with and without internal CO2 level control. Average curves of baseline-subtracted fluorescence values of 48 replicates are plotted.

Evaluation of the toxic effects of PFOS in hESCs differentiating into cardiomyocytes

In this application note, R. Yang, et.al. studied the effects of an environmental toxin, PFOS, on the differentiation of hESCs into cardiomyocytes as a model for understanding the impact of PFOS exposure on heart development during pregnancy.

We demonstrate the benefits of using the Varioskan LUX Multimode Microplate Reader and the EVOS FL Imaging System to detect changes in cell viability and mitochondrial membrane potential (MMP) of differentiating hESCs upon treatment with PFOS.

Cell viability on differentiation days 2, 4, and 8 after chemical treatment was measured with Invitrogen PrestoBlue HS Cell Viability Reagent, a one-step resazurin-based cell viability assay, which is a cost-effective, sensitive, and simple method to detect cellular viability. PFOS has been shown to cause mitochondrial toxicity and depolarization of the mitochondrial membrane. Thus, we measured MMP by staining the differentiating cardiac cells with JC-1 dye (part of the Invitrogen MitoProbe JC-1 Assay Kit) after PFOS treatment.

Schematic diagram of experimental approach for evaluating potential PFOS toxicity, showing the steps from cell culture to treatment to cell imaging
Figure 3. Experimental layout for evaluation of potential developmental cardiac toxicity of PFOS. hESCs were seeded as single cells in 96-well plates and differentiated into beating cardiomyocytes for 8 days, as indicated. Cell viability and MMP were detected with the Varioskan LUX Multimode Microplate Reader and EVOS FL Imaging System. In the schematic of the 96-well plate, blue wells are filled with DPBS (the wells from B10 to G10 were filled with CDM3 medium only prior to cell viability measurements); white wells are filled with cells treated with DMSO; orange wells are filled with cells treated with different concentrations of PFOS.

FluoroSpot Pre-Screening with Thermo Scientific Microplate Readers

Thumbnail of page 1 of web-based app note: FluoroSpot Pre-Screening with Thermo Scientific Microplate Readers

In this technical note, we introduce a new image-independent procedure for the initial evaluation of FluoroSpot plates, which can be performed with Thermo Scientific fluorescence microplate readers (Varioskan LUX Multimode Microplate Reader, Fluoroskan Microplate Fluorometer or Fluoroskan FL Microplate Fluorometer and Luminometer). For the current study, FluoroSpot assays were evaluated using the Varioskan LUX multimode reader. The procedure involves a rapid measurement of the fluorescence intensity within a defined arrangement of multiple points on the bottom of every well. The limit of detection (LOD) of this FluoroSpot pre-screening is calculated here for two model cytokines (IFN-γ and IL-2). The limitations and applicability of this image-independent FluoroSpot pre-screening method are also further discussed.

ELISpot and FluoroSpot assay workflow represented as simplified five-step graphic concluding with both brightfield (ELISpot) and fluorescence (FluoroSpot) images of results
Figure 4. The ELISpot/FluoroSpot assay workflow.

Monitoring cell health with alamarBlue and PrestoBlue reagents using the Varioskan LUX Multimode Microplate Reader

In this application note, the benefits of pairing alamarBlue and PrestoBlue reagents with the Varioskan LUX Multimode Microplate Reader are demonstrated. Both viability assays are simple to perform, and the generated signal (fluorescence or absorbance) typically can be read in a conventional microplate reader. However, utilizing the Varioskan LUX Multimode Microplate Reader and SkanIt Software significantly enhances the usability of these resazurin-based workflows (Figure 1), allowing fast readout of signals and immediate access to powerful data processing steps such as curve fitting and useful calculations, including cell viability percentages and cytotoxic potency.

Black microplate with clear-bottom wells treated with alamarBlue or PrestoBlue reagent exhibiting a rainbow-style coloring going left to right green-blue-purple
Screenshot in SkanIt Software of the heatmap data visualization of the same microplate from (A), read using fluorescence mode of the Varioskan LUX multimode reader

Figure 5. Cell health monitoring with alamarBlue and PrestoBluereagents. (A) Plate and (B) SkanIt Software data visualization.

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Absorbance
Plate types 6, 384 well plates
Wavelength selection Double monochromators
Wavelength range 200–1,000 nm
Light source Xenon flash lamp
Read out range 0–6 Abs
Linear measurement range 0–4 Abs (96-well plate) at 450 nm, ± 2%
0–3 Abs (384-well plate) at 450 nm, ± 2%
Accuracy 0.003 Abs or ± 2%, at 200–399 nm (0–2 Abs)
0.003 Abs or ± 1%, at 400–1,000 nm (0–3 Abs)
Precision SD <0.001 Abs or CV <0.5%, at 450 nm (0–3 Abs)
Fluorescence Intensity
Plate types 6–1,536 well plates
Wavelength selection  Double excitation and emission monochromators
Excitation wavelength range 200–1,000 nm
Emission wavelength range  270–840 nm
Light source Xenon flash lamp
Sensitivity Top reading: <0.4 fmol fluorescein/well (black 384-well plate)
Bottom reading: <4 fmol fluorescein/well, (clear bottom black 384 square well plate)
Dynamic range Top reading >6 decades
Bottom reading >5.5 decades
Time-resolved fluorescence
Plate types 6–1,536 well plates
Wavelength selection Filters (spectral scanning with double excitation and emission monochromators)
Excitation wavelength range Fixed to 334 nm (spectral scanning 200–840 nm)
Emission wavelength range 400–700 nm (spectral scanning 270–840 nm)
Light source  Xenon flash lamp
Sensitivity <1 amol Eu/well (white low volume 384 well plate)
Dynamic range >6 decades
Luminescence
Plate types 6–1,536 well plates (spectral scanning 6, 384 well plates)
Wavelength selection Direct or filters (spectral scanning with double monochromators)
Wavelength range  360–670 nm
Sensitivity <7 amol ATP/well (white 384-well plate)
Dynamic range >7 decades
AlphaScreen
Plate types 6, 1,536 well plates
Wavelength selection  Filters
Excitation wavelength range Fixed to 680 nm
Emission wavelength range 400–660 nm
Light source  LED
Sensitivity <100 amol phosphotyrosine/well (white 384-well plate)
Dispensing
Plate types 6, 384 well plates
No. of dispensers None, one or two
Syringe size  1 ml (standard), 5 ml (optional)
Dispensing volume 2–5,000 μl, in 1 µl increments (1 ml syringe)
5–25,000 μl, in 5 µl increments (5 ml syringe)
Accuracy <1 μl with 50 μl (0.4 mm tip)
<0.2 μl with 5 μl (0.25 mm tip)
Precision <1 μl with 50 μl (0.4 mm tip)
<0.25 μl with 5 μl (0.25 mm tip)
Dead volume Reagent loss <100 μl, Total tubing volume <800 μl
Incubator and Shaker
Temperature range From ambient + 4°C to 45 °C
Shaking type  Orbital
Integrated Gas Module
CO2 concentration range 0.1–15%
CO2 concentration stability  ± 0.3% at 5% CO2
O2 concentration range 1–21 %
O2 concentration stability  ±0.3% at 1% O2
General Features
Measurement modes End-point, kinetic, spectra, multipoint and kinetic spectra
Measurement speed Reads a 96-well plate in 15 s, a 384-well plate in 45 s, and a 1,536-well plate in 135 s (minimum times)
Interface  PC software (SkanIt software)
Dimensions (D x W x H) 58 x 53 x 51 cm (23 x 21 x 20 in)
Weight  54–59 kg (119–130 lb), depending on configuration

Low-volume quantification of nucleic acids and proteins with Thermo Scientific µDrop and µDrop Duo Plates

Introduction

Photometry in the UV range is a common way to quantify nucleic acids and proteins in a sample. Both nucleic acids and proteins absorb UV light. In the case of nucleic acids, the nitrogenous bases present in all nucleotides have a clearly distinguishable absorption maximum at 260 nm. On the other hand, in the case of proteins, the light absorptive properties at 280 nm are limited to few amino acids, specifically the aromatic amino acids tryptophan (Trp) and tyrosine (Tyr), and to a lesser extent cysteine groups forming disulfide bonds (Cys-Cys). Therefore, the absorption of proteins and peptides at 280 nm is proportional to the content of these amino acids.

These UV-absorptive properties make it possible to use direct photometry to quantify the concentrations of nucleic acids and proteins. Such calculations are performed using Beer-Lambert’s Law, which describes that the absorbance of a certain nucleic acid or a protein depends on the molecule’s concentration, the molecule’s absorptivity coefficient (given by the intrinsic absorptivity properties of such molecule) and the pathlength of the incident light. The use of this equation specifically for calculation of nucleic acids and proteins is shown on Table 1.

Table 1. Calculation of nucleic acids and protein concentrations using direct absorbance measurements, according to Beer-Lambert’s Law.

  Nucleic acids Proteins
Example case Double stranded DNA (dsDNA) Bovine serum albumin (BSA)
Equation for the calculations

Where:
C = concentration of dsDNA (µg/mL)
A260 = absorbance value at 260 nm of sample and blank
A320 = absorbance value at 320 nm of sample and blank
L = light pathlength (cm)
εdsDNA = extinction coefficient of dsDNA = 0.02 (µg/mL)-1cm-1
Note: Ratio of is 50 (µg/mL) cm

Where:
C = concentration of BSA (mg/mL)
A280 = absorbance value at 260 nm of sample and blank
A320 = absorbance value at 320 nm of sample and blank
L = light pathlength (cm)
ε1% = mass extinction coefficient of BSA = 6.7 (mg/mL)-1cm-1 for 1% (10 mg/mL) BSA solution

Equation for molar extinction coefficient for ε proteins   Molar extinction coefficient ε for any protein can be easily calculated using following equation:
ε=(nW * 5500) + (nY * 1490) + (nC * 125)
Where:
W: tryptophan, Y: tyrosine, C: cysteine
n: number of each residue present in the protein
(5500, 1490, and 125: molar absorptivity at 280 nm of W, Y, and C, respectively)

The calculations of nucleic acids and proteins are performed based upon absorbance measurements at 260 nm and 280 nm, respectively, but an additional measurement at 320 nm is typically carried out with the purpose of background subtraction. Measuring at 320 nm allows excluding impurities in the sample, for example arising from the presence of magnetic beads in the sample.
 

Low-volume quantification with Thermo Scientific µDrop Plate and µDrop Duo Plates

The amount of sample available for nucleic acid and protein analysis is often low. To address that challenge, Thermo Scientific offers the µDrop and µDrop Duo Plates, which enable direct photometric measurements at a microliter scale (Figure 1). The Drop and µDrop Duo Plates consist of two separate measurement locations: the left side is intended for measurements of low-sample volumes and the right side is for measuring cuvettes. The cuvette slot is used to perform photometric measurements (including kinetic studies) with standard cuvettes that are covered with stopper and placed in a horizontal position.

The low-volume measurement area consists of two or four quartz slides: one or two pairs of the top clear quartz slide and the bottom quartz slide, which is partially Teflon-coated. The bottom slide contains a matrix array position where samples are pipetted. In the case of the µDrop Plate, there are 16 samples positions arranged in a 2 x 8 matrix, while in the case of the µDrop Duo Plate, there are 32 sample positions arranged in 2 separated arrays also in a 2 x 8 matrix (Figure 1). The pathlength of the µDrop Plate is fixed, and the nominal value is 0.05 cm. However, the pathlength can vary between 0.048 and 0.052 cm, and verified pathlength for each μDrop Plate is indicated on the quality control measurement report delivered with the μDrop Plate. Same applies to the µDrop Duo Plate (with 32 samples), but in this case, there are two separate pathlength values, one for each 2 x 8 array. These exact pathlength values should always be used for the concentration calculations, instead of the nominal value of 0.05 cm, in order to obtain more accurate results.

top view of the plates showing the quartz slides and the positions for pipetted samples

Figure 1. μDrop Plate and the μDrop Duo Plate.

Compared to a normal cuvette having a pathlength of 1 cm, the pathlength of the μDrop Plate or μDrop Duo Plate is 20 times shorter (0.05 cm). Because of this, there is roughly a 20 times difference between the concentrations that can be measured with a typical cuvette versus the μDrop and μDrop Duo Plate. This means that the detection limit of any given analyte is about 20 times higher in the μDrop and μDrop Duo Plate than with cuvettes, when measured in the same instrument. On the other hand, a shorter pathlength allows for lower sample volumes to be measured. In the case of the μDrop and μDrop Duo Plate, a sample volume down to 2 µL can be used. It is possible to measure nucleic acid and protein concentrations from a few to thousands of micrograms per microliter using these devices together with a photometer that has sufficiently high precision and a wide linear range, such as the Thermo Scientific Multiskan SkyHigh Microplate Spectrophotometer. Finally, any photometric measurement device always has a certain background absorption. Therefore, blank subtraction is necessary when photometric quantification of the sample concentrations is performed.


Materials and methods

To calculate the detection limits for nucleic acid quantification (dsDNA), 11 dilutions were made of a DNA stock solution of Herring sperm DNA (Promega, D1816) using TE-buffer, (Invitrogen 10 mM Tris-HCl, 0.1 mM EDTA), to cover concentration range between 50 and 3500 µg/mL. To calculate the detection limits for protein quantification (BSA), 9 dilutions were made of a BSA stock solution (Sigma, A7030) to distilled water to cover concentration range between 0.5 and 100 mg/ml. The blanks as well as dsDNA or BSA samples were measured by pipetting 4 µL into the measurement areas of the μDrop Plate and the µDrop Duo Plate. In case of both μDrop and µDrop duo plates, 2 blanks were used. All measurements were performed using Multiskan SkyHigh Microplate Spectrophotometer with touchscreen and cuvette.

As indicated earlier, to ensure that proper concentration calculations, the exact pathlength values of the μDrop Plate or µDrop Duo Plate need to be provided. These values were defined in Multiskan SkyHigh settings by selecting “Settings” from the Home screen and then selecting “μDrop Plates” as indicated in Figure 2. The final concentrations reported by the instrument will be calculated with these pathlength values, and the user will not need to perform any correction steps, afterwards. Ready-made assay protocols in the Multiskan SkyHigh user interface for dsDNA or BSA quantification were used to perform the assays.

User interface screenshot showing how users specify pathlengths into the software

Figure 2. Setting-up the pathlength values of the μDrop Plate and μDrop Duo Plate in the User Interface of the Multiskan SkyHigh Microplate Spectrophotometer.

When the Multiskan SkyHigh model without touch screen is used, the dsDNA concentration calculations need to be done using the Thermo Scientific SkanIt software, which controls all the Thermo Scientific microplate readers. For that purpose, in “Plate Layout”, the appropriate plate format should be selected from the dropdown menu (μDrop Plate or μDrop Duo Plate). In the case of the μDrop Duo Plate, it is possible to add two values as the pathlengths on the two separate measurement areas may differ. See an example of the performed calculations in Figure 3.

Screenshot of software showing the results of the dsDNA concentration calculations

Figure 3. Setting-up the pathlength values of the μDrop Duo Plate in the SkanIt software for calculations of the dsDNA concentrations using Multiskan SkyHigh Microplate Spectrophotometer. If the calculation is performed without 320 nm subtraction, the factor 50 is simply added to the pathlength correction step of the 260 nm measurement data.

Multiskan SkyHigh models with UI, the ready-made sessions have all the necessary calculations. The results automatically contain the concentration, purity ratios and the sample spectrum, Figure 4 below.

Calculations results screen showing the dsDNA measurements and corresponding concentration and purity values

Figure 4. An example data set of a µDrop duo plate dsDNA measurement.


Results and discussion

Detection range of the µDrop Plate and µDrop Duo Plates

The detection range is given by the lowest and highest possible concentrations that can be reliably measured with a given instrument. Therefore, these two extremes of the detection range are instrument dependent and they can be theoretically calculated from the instrument’s performance specifications. In this note, however, we focus on the detection ranges of nucleic acids and proteins, as experimentally measured using IUPAC recommendations. Often the limiting factor is the lowest end of the detection range, since unknown samples that have too low concentrations of nucleic acids or proteins (falling under the lowest part of the detection range) cannot be simply accurately estimated using photometric detection with the µDrop Plate or µDrop Duo Plates. Often, because most microplate spectrophotometers in the market have very similar detection ranges, the only solution in such cases is to change the detection technology and use instead fluorescence-based detection, which is known to be considerably more sensitive. By contrast, for samples with too high concentration values (falling outside of the detection range), a simple solution is to dilute the samples, so that they can fall within the detection range, and thus can be accurately measured.

Limits of Detection (LOD) and Limits of Quantification (LOQ) for measuring nucleic acids and proteins

The lowest part of the detection range curve indicates the assay sensitivity, which can be assessed, according to the IUPAC, with two different parameters: Limit of Detection (LOD) and Limit of Quantification (LOQ). The LOD is defined as the lowest quantity or concentration of analyte that can be separated from the background or blank values. The LOD value means that the presence of the analyte can be detected with statistical significance, but the analyte cannot be quantified as an exact value. On the other hand, the LOQ is defined as the lowest quantity or concentration of the analyte at which quantification is possible with statistical relevance. In practice, LOD is the limiting value in qualitative assays where the question “is there any analyte in my sample or not?” needs to be answered. In such cases, LOQ is the limiting value that the user can measure as the concentration of the analyte in quantitative assays. Both LOD and LOQ calculations assume that both blank samples and replicates of the analyte are normally distributed. Signals at the LOD are defined as the blank mean + 3 * standard deviation of the blank, while signals at the LOQ are defined as blank mean + 10 * standard deviation of the blanks. Experimental calculations of the LOD and LOQ values is detailed in Table 2.

Table 2. Mathematical definitions of Limits of Detection (LOD) and Limits of Quantification (LOQ).

  LOD LOQ
Equations for the calculations

 

Where:
k = 3
SD blanks = standard deviations of instrument readings taken on assay blanks
m = slope of a graph of instrument’s blanked signal vs. concentration of the analyte on the linear range, as calculated by linear regression

 

Where:
k = 10
SD blanks = standard deviations of instrument readings taken on assay blanks
m = slope of a graph of instrument’s blanked signal vs. concentration of the analyte on the linear range, as calculated by linear regression

The calculated values for LOD and LOQ for both nucleic acids and proteins, exemplified with dsDNA and BSA are reported in Table 3.

Table 3. Limits of Detection (LOD) and Limits of Quantification (LOQ) calculated for nucleic acids (dsDNA) and proteins (BSA) using the µDrop Plate or µDrop Duo Plates.

    µDrop Plate µDrop Duo Plate
Nucleic acid (dsDNA) LOD 2.0 µg/mL* 2.9 µg/mL*
LOQ 6.5 µg/mL * 9.8 µg/mL *
Protein (BSA) LOD 0.4 mg/ml 0.4 mg/ml
LOQ 1.5 mg/ml 1.5 mg/ml

*This concentration difference between µDrop and µDrop Duo plate is statistically meaningless because it is caused by random approx. 0.001 Abs difference in the measured Abs 260 nm value. This A260 nm difference is below the precision specification of the instrument and therefore the difference in LOD and LOQ values between these two µDrop plate types is individual random happening in this test.


Linearity ranges

The dsDNA experiment described above is also used as an example to demonstrate the linear range of the Multiskan SkyHigh and µDrop plate system in practice.

As mentioned in the detection range section, with a measurement device with fixed pathlength, like the µDrop plates, the maximum measurement range is always determined by the instrument. The lower part is determined by the precision of the blank (LOD) and the upper part by the linear range of the instrument. So, for Multiskan SkyHigh, which is specified to be linear up to 2.5 Abs, the theoretical maximum concentration is 2.5 x 50 μg/ml / 0.050= 2500 μg/ml

The absorbance values of both µDrop and µDrop Duo plates are plotted as function of the theoretical concentration of the dsDNA samples in Figure 5.

Graph of absorbance vs concentration for µDrop and µDrop Duo plates compared to theoretical concentrations of sample

Figure 5. µDrop and µDrop Duo plate absorbance A260 nm values as function of the theoretical dsDNA concentration.

This data shows that the linear range up to 2.5 Abs well applies also to Multiskan SkyHigh with the µDrop plates in dsDNA A260 nm measurements.


Conclusions

Multiskan SkyHigh combined with μDrop or μDrop Duo Plate is an ideal tool for quick and easy concentration measurements of DNA, RNA, protein samples, or spectral scanning with very low sample consumption. Multiskan SkyHigh, µDrop plates and ready-made sessions for nucleic acid and protein analysis in the UI and in the SkanIt cloud library offer a very easy to use approach to these measurements. Samples are easy to pipette onto the μDrop Plates with a single or an 8-channel pipette. The plates are easily wiped clean, making it convenient to be used in serial measurements.

Read about the µDrop and µDrop Duo plates

Q: What instrument features can I add later as an upgrade?

A: Absorbance and fluorescence intensity are included with every instrument. The measurement technologies of the LAT module (Luminescence, AlphaScreen and Time-Resolved Fluorescence) can be upgraded. The only limitation is that the LAT module cannot exist without luminescence technology. Thus, if the researcher wants AlphaScreen and/or time-resolved fluorescence, they will also get luminescence. The dispensers and integrated gas module are also upgradable. Bottom reading, however, must be included in the original order, and cannot be added later.

Q: For what measurement technologies do I need to purchase and install filters?

A: In time-resolved fluorescence and AlphaScreen the instrument requires filters for wavelength selection. The excitation filters for both technologies are built-in, but the emission filters must be ordered and installed separately. When you install filters to the LAT module, you have to enter the filter information to the instrument via SkanIt software. Absorbance and fluorescence intensity technologies use monochromators for wavelength selection, thus filters are not necessary. Most of the luminescence assays do not require any wavelength selection. But if required, filters can be installed.

Q: What measurement technologies allow spectral scanning?

A: The instrument includes monochromators to allow spectral scanning in all measurement technologies except AlphaScreen. The instrument must have the corresponding measurement technology onboard to be able to run spectral scanning.

Q: How do I choose the right plate adapter?

A: The plate adapter lifts the microplate to optimal height for measurement and dispensing. Choose the adapter based on the plate format (96-well, 384-well, etc.) and whether or not the plates have lids. When starting a measurement, the SkanIt software checks that the installed plate adapter is compatible with the plate template selected, and informs you if there’s a mismatch.

Q: When should I use bottom reading?

A: Bottom reading should be used when measuring adherent cells that are at the bottom of the well. From the bottom side the excitation light can be focused more accurately on the cell layer. Bottom reading is available only in fluorescence intensity measurements with an instrument that has the bottom reading option.

Q: There are two dispensing positions in the instrument: F and L. How do I choose the right one?

A: Either position can be used with either dispenser. If the assay reaction is fast and the signal changes quickly after triggering the reaction by dispensing, select the dispensing position which points at the measurement position of the technology you will measure. If you use a dispensing position that does not point at the correct measurement position, the instrument makes an extra plate movement between dispensing and measurement. See Varioskan LUX user manual for more information on which measurement technologies match with dispensing position F or position L.

Q: Varioskan LUX is able to dispense and measure simultaneously. In what type of assay is this important?

A: It is important in fast kinetic assays where the peak signal is reached soon after reaction triggering. If the peak signal is reached in less than a second after dispensing, for example, simultaneous dispensing and measurement allows you to follow the signal progress from the very start of the reaction without losing the signal peak.

Q: How does Varioskan LUX incubator prevent condensation from forming on the microplate lid, and why is it important?

A: Condensation on the microplate lid affects measurement results. In the Varioskan LUX, the microplate is surrounded by temperature-controlled heaters. The upper element is slightly warmer than the lower element to avoid condensation on the plate lid.

Q: Can I install SkanIt software on several computers?

A: Yes, there is no limit on software licenses within the same customer organization. Measurement session files can even be exported and imported between SkanIt software installed on different computers, allowing researchers to work with the software outside the laboratory.

Q: How does the Drug Discovery Edition of SkanIt software differ from the Research Edition?

A: The Drug Discovery Edition offers features required for compliancy with FDA 21 CFR Part 11, a must for the pharma industry. Those special software features provide an audit trail, require an electronic signature, and a prompt to provide a reason for any changes made. Apart from these three special features, the Research Edition and the Drug Discovery Edition have the same features and functionalities.

Q: If I abort the run before it’s finished, does the measurement data disappear?

A: No, the data up to the time of abortion is saved. SkanIt software saves the measurement data automatically and constantly to the software database during a run. If a long kinetic measurement is aborted either unexpectedly or intentionally, the data does not disappear.

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Available measurement technology configurations

All configurations available with top or top and bottom reading; none, one, or two dispensers; and optional integrated gas modules.

System configuration options
Absorbance and Fluorescence intensity
Absorbance, Fluorescence intensity, and Luminescence
Absorbance, Fluorescence intensity, Luminescence, and AlphaScreen
Absorbance, Fluorescence intensity, Luminescence, and Time-resolved fluorescence  
Absorbance, Fluorescence intensity, Luminescence, Time-resolved fluorescence, and AlphaScreen

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