Counting and Standard Slides, electronic records, light cubes, and fluorescent stains for Countess Automated Cell Counters

Evaluate the options between convenient disposable slides, economical reusable slides, and our functional standard slide.  A new software module supports electronic record-keeping compliance. For the Countess 3 FL Automated Cell Counter, EVOS light cubes provide constant, brilliant illumination, while ReadyCount reagents are optimized for fluorescence counting applications.

Slides and slide holders

With both the Countess 3 and Countess 3 FL Automated Cell Counters, you can choose between convenient disposable slides and economical, environmentally-friendly reusable slides. Both are designed specifically for use with Countess cell counters.

 

Each slide has two chambers to count one sample in duplicate or two different samples. Only 10 µL of sample mixed with 10 µL of trypan blue is needed for a brightfield count. Cells are counted in a central location of the counting chamber. The sample volume counted is 0.4 µL, equivalent to four 1-mm² squares in a standard hemocytometer.

Disposable Countess Cell Counting Chamber Slides are convenient for quickly processing cell samples when there are many to process, when the cell culture facility is busy, or to simplify the disposal of contaminated or hazardous samples. Disposable slides also eliminate the risk of contamination from residual samples and the frustration of finding an unclean shared hemocytometer. Unlike a hemocytometer, there is no need to clean Chamber slides before or after use.

 

The Countess Reusable Slide is a green, cost-efficient alternative to disposable slides. Even if your lab uses only a few slides per day, you can reduce cost and generate less waste per year with the reusable slide. This innovation makes it affordable for labs to automate their cell counts. The reusable slide should be cleaned and dried between counts, just as with a hemocytometer.

The Chamber slide and Reusable slide have different slide holders. When using the Chamber slides, the holder can remain in the counter so that you can easily insert each slide directly into it. The reusable slide is inserted into the slide holder first, then the combination of slide and holder are inserted into the Countess 3 or 3 FL Automated Cell Counter.

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Countess 3 Standard Slide

Use the Countess 3 Standard Slide as a functional reference for viability and cell counts. This ready-to-use slide contains between 0.5 and 1.5 x 10⁶/mL permanently mounted 15 µm microspheres in a cell counting chamber. When viewed in brightfield on the Countess 3 or FL Automated Cell Counter, the microspheres mimic the appearance of a 1:1 mixture of live and dead cells stained with trypan blue solution.  

Countess 3 Standard Slide in brightfield mode

Viewed in brightfield mode, the microspheres on the Standard Slide mimic approximately 50% live and 50% dead cells with a concentration between 0.5 and 1.5 x 10⁶ cells/mL.

 

Countess 3 Standard Slide in fluorescence mode

Viewed in fluorescence mode, the microspheres on the Standard Slide mimic approximately 50% blue/green and 50% red cells with a concentration between 0.5 and 1.5 x 10⁶ cells/mL The blue/green spheres can be viewed with a GFP or DAPI light cube and the red spheres with RFP, Texas Red, or Cy5. A small percentage of spheres will fluoresce in both colors.

When viewed on the Countess 3 FL Automated Cell Counter, the microspheres also are fluorescent in a 1:1 mixture, providing approximately 50% detection with a DAPI or GFP light cube and approximately 50% detection with an RFP, Texas Red, or Cy5 light cube. The 50% fluorescence populations are shown in both single- and dual-channel fluorescence counting.

 

The permanently mounted microspheres on the Countess 3 Standard Slide will have minimal variability from one count to the next. You can use the slide as a functional reference for cell counts and viability across days or between Countess 3 instruments. The slide is ready to use and reusable.

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21 CFR Part 11 compliance support software

The Countess SAE Software Solution is an optional software module that supports electronic record-keeping that is compliant with the US Food and Drug Administration’s Electronic Records and Signatures Rule (21 CFR Part 11). Capabilities of the software include restriction of unauthorized users, password policies and expiration dates, defined user permissions and roles, tracking of all data changes through an audit log, audit reports, and on-board e-signature workflows. All Countess 3 and Countess 3 FL Automated Cell Counters can run in SAE Mode after purchase of the Countess SAE Software License. 

 

For more information about the Countess SAE Software Solution, see Features.

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To use the Countess SAE Software Solution with your Countess 3 Automated Cell Counter, a software license can be purchased independently or as part of a bundled Countess 3 FL instrument and software license package.

EVOS light cubes

With the fluorescence capabilities of the Countess 3 FL Automated Cell Counter, you can easily visualize fluorescent proteins and perform apoptosis or proliferation assays in your cell culture room. 

 

The Invitrogen EVOS LED Light Cubes that power these fluorescence applications on the Countess 3 FL provide more than 50,000 hours of brilliant (yet adjustable) illumination that remains constant with minimal degradation over time. Cost-efficient, environmentally friendly, and spectrally optimized, EVOS LED light cubes are designed for precise control, minimal maintenance, and exceptional reliability.

 

Each cube contains an LED, illuminating optics, and filters. Light cubes are user-interchangeable and automatically detected by the Countess 3 FL system for plug-and-play ease.

 

Tip: Light cubes are not included with the Countess 3 FL cell counter to allow customization based on dyes and fluorescent proteins used. 

 

Choose light cubes with spectra that match the dyes and counting applications you want to use. Light cubes are easy to swap in and out and can be purchased at any time to meet new application requirements. A wide range of dyes is supported.

 

Although many researchers choose GFP and RFP for fluorescent protein detection, there are 14 different EVOS LED Light Cube choices. DAPI is a traditionally used nuclear dye. The table shows common dyes detected by seven popular light cubes.

Seven popular light cubes and their common compatible dyes

Cat. no.  Light cube  Excitation (nm)  Emission (nm)  Common compatible dyes/fluorescent proteins 
AMEP4950 DAPI 2.0  357/44  447/60  DAPI, Hoechst, NucBlue, Alexa Fluor 350, BFP 
AMEP4951 GFP 2.0  470/22  525/50  GFP, Alexa Fluor 488, SYBR Green, FITC, Calcein, Fluo-4, MitoTracker Green, pHrodo Green 
AMEP4954 YFP 2.0 500/24  542/27  EYFP, acridine orange (+DNA) 
AMEP4952  RFP 2.0  531/40  593/40  RFP, Alexa Fluor 546, Alexa Fluor 555, Cy3, PE, dTomato, MitoTracker Orange, Rhodamine Red, SYTOX Orange 
AMEP4955  Texas Red 2.0  585/29  628/32  Texas Red, Alex Fluor 568, Alexa Fluor 594, MitoTracker CMXRos, mCherry, pHrodo Red, Cy3.5, CellTracker Red CMTPX 
AMEP4956 Cy5 2.0  628/40  692/40  Cy5, Alexa Fluor 647, Alexa Fluor660, DRAQ5 
AMEP4667  Cy7  710/40  809/81  Cy7, IRDye™ 800CW, DyLight 750 

ReadyCount reagents

Fluorescence staining can provide rich data on cells, especially in heterogeneous samples, but commercial stains can be difficult to mix, titrate, and apply for optimal performance. Invitrogen ReadyCount reagents are bench-stable fluorescent stains that are pre-optimized for use with Countess 3 FL Automated Cell Counter. 

 

ReadyCount reagents can be used to stain specific nucleated cells within mixed populations, reducing complexity and improving count-to-count and sample-to-sample reproducibility when matched to the appropriate EVOS light cube. Simply mix 10 µL of cell sample with 10 µL of stain, pipet the solution into a Countess chamber or reusable slide, and insert the slide into the instrument.

ReadyCount Red Dead Cell Stain stains dead cells and will fluoresce when used with an EVOS RFP or Texas Red light cube

ReadyCount Blue Nuclear Stain stains nucleic acids and will fluoresce when used with an EVOS DAPI light cube.

ReadyCount Green/Red Cell Viability Stain is an optimized AO/PI mixture that stains nucleated cells green and dead cells red, and will fluoresce and be counted when used with EVOS GFP and RFP or Texas Red light cubes. Cells are shown at (left) 1.0x magnification and (right) 4.0x magnification.

For example, ReadyCount Green/Red Cell Viability Stain is an optimized mix of acridine orange and propidium iodide (AO/PI), which are standard stains for nucleated cells and viability, respectively. Even in peripheral blood mononuclear cell (PBMC) samples, which are notoriously difficult to count using trypan blue, AO/PI stains allow the discrimination of red blood cells (RBCs), platelets, and debris.

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