Enhanced Alexa Fluor secondary antibodies: Higher signal-to-noise ratios for hard-to-detect targets

Invitrogen Alexa Fluor Plus secondary antibodies are designed to help you detect low-abundance targets in rare or critical samples using fluorescence imaging or western blotting. The Alexa Fluor antibodies you rely on for superior brightness and photostability are now available in an exclusive formulation using proprietary “plus” dye chemistry, which provides up to 4 times higher signal-to-noise ratios. This unique formulation, coupled with stringent pre-adsorption protocols to remove antibodies that bind nonspecifically, enables Alexa Fluor Plus antibodies to offer enhanced sensitivity and minimal cross-reactivity for those challenging samples.

 

Improved signal-to-noise ratios with Alexa Fluor Plus antibodies reveal greater detail. Fixed and permeabilized E18 Sprague Dawley™ primary cortical neuronal cells were labeled with anti–β-III tubulin primary antibody and (A) Invitrogen Alexa Fluor 488 or (B) Invitrogen Alexa Fluor Plus 488 secondary antibodies. Cells were then counterstained with Hoechst 33342 dye and imaged on a Zeiss LSM 710 inverted confocal microscope at 40x magnification.


Improved formulation for Pierce protease and phosphatase inhibitor tablets

Thermo Scientific Pierce protease and phosphatase inhibitor tablets are ideal for the protection of proteins during extraction procedures or lysate preparation using primary cells, cultured mammalian cells, animal tissues, plant tissues, yeast cells, or bacterial cells. With improved formulations, these ready-to-use inhibitor tablets dissolve quickly into a clear solution and are directly compatible with Thermo Scientific Pierce BCA Protein Assays.

 

Performance comparison between three commercially available protease inhibitor tablets. Pancreatic extract (100 μL, 0.5 μg/μL) was incubated with cleavable fluorogenic substrates for trypsin and cysteine proteases, metalloproteases, and cathepsins, in the presence of Thermo Scientific Pierce Protease Inhibitor Mini Tablets, Roche cOmplete™ Protease Inhibitor Tablets, and Sigma- Aldrich SIGMAFAST™ Protease Inhibitor Cocktail Tablets, with and without EDTA. Reactions were incubated for 1 hr at 37ºC, and fluorescence was determined at the appropriate detection emissions on a Thermo Scientific Varioskan Flash microplate reader. Percent protease inhibition is shown for each protease inhibitor formulation.


New and improved Tris-glycine midi protein gels

Invitrogen Novex Tris-Glycine Plus Midi Gels are the next generation of precast midi polyacrylamide gels, providing high-quality performance and separation of proteins into wellresolved bands using Laemmli electrophoresis technology. Available in fixed (10%, 12%) and gradient (4–12%, 4–20%, 8–16%) concentrations and with different well formats (12+2 wells, 20 wells, 26 wells), these Tris-glycine gels feature a longer shelf life (up to 1 year at 4°C), fast run conditions (<60 minutes at constant voltage), and the flexibility to run native or denatured protein samples.

 

 

 

Novex Tris-Glycine Plus Midi Gel System.


Improvements to the alamarBlue cell viability and cytotoxicity reagent

For over 50 years, the alamarBlue dye has proven to be a reliable reagent for measuring cell viability in bacteria, yeast, fungi, protozoa, and cultured mammalian cells. This resazurin-based redox indicator is converted into a fluorescent and colorimetric product (resorufin) by metabolically active cells. Damaged and nonviable cells have lower innate metabolic activity and generate proportionally lower signals.

The Invitrogen alamarBlue Cell Viability Reagent is a mixture of salts, a background suppressor, and resazurin—a formulation that accurately and reliably reports changes in redox potential. Because of the highly sensitive nature of the alamarBlue reagent, the manufacturing processes need to be well controlled. In the absence of proper manufacturing processes, the background fluorescence signal of the reagent could increase, causing a decrease in the signal-to-background ratio and a reduction in assay performance. Our recently improved manufacturing process has resulted in a product with superior performance, displaying >50% reduction in background fluorescence compared with the previously manufactured material or another commercially available product.

 

Evaluation of our new manufacturing process for the alamarBlue reagent. (A) Comparison of the background fluorescence from various alamarBlue products reveals that the improved Invitrogen alamarBlue Cell Viability Reagent shows the lowest level of background signal. (B) As expected, the decrease in background fluorescence results in an increase in the signal-tonoise ratio. The newly manufactured alamarBlue reagent has a significantly higher signal-to-background ratio than the previously manufactured reagent or another commercially available product.


Introducing the FluxOR Red Potassium Ion Channel Assay

The Invitrogen FluxOR Red Potassium Ion Channel Assay utilizes a red-fluorescent indicator dye to detect potassium ion channel activity. Similar to the Invitrogen FluxOR II Green assay, the FluxOR Red assay takes advantage of the well-established permeability of potassium channels to thallium ions. When potassium channels are opened by a stimulus, extracellular thallium flows down its concentration gradient into cells and binds to the FluxOR thallium-sensitive dye. In the FluxOR Red assay, an increase in cytosolic red fluorescence (Ex/Em = 560/590 nm) serves as a surrogate measurement of the activity of any ion channel or transport process that allows thallium into cells. The FluxOR Red Potassium Ion Channel Assay can be configured for either imaging or high-throughput screening applications and is compatible with detection of GFP and other green-fluorescent probes including the FluxOR II Green dye, which can be used as a second, independent measure of potassium channel activity.

 

Multiplexing the FluxOR Red Potassium Ion Channel Assay with a cell viability assay. Gibco Primary Rat Cortex Neurons (Cat. No. A1084001) were incubated with Invitrogen Calcein AM and Invitrogen FluxOR Red dye. After stimulation of the potassium channels, cells were imaged on the Invitrogen EVOS FL Auto 2 Imaging System using (A) a FITC/Alexa Fluor 488 filter set to detect the green fluorescence of live cells stained with calcein and (B) a TRITC/Alexa Fluor 555 filter set to detect the red fluorescence of the FluxOR Red reagent, indicative of potassium ion flux. (C) The overlay of the calcein and FluxOR staining images confirms that live cells show potassium channel activity.


Follow LDL uptake and trafficking using pHrodo Red and pHrodo Green LDL conjugates

A key lipid–protein complex in blood, the human low-density lipoprotein (LDL) is a crucial component of metabolism that drives the transport of fats throughout the body. It is now possible to follow—in real time—the receptor-mediated endocytosis and trafficking of the LDL complex in live cells using Invitrogen pHrodo Red and pHrodo Green LDL conjugates. Though dimly fluorescent at neutral pH outside of cells, pHrodo LDL conjugates fluoresce brightly during and after internalization as endosomes are increasingly acidified through the endocytic pathway.

With a simple workflow (add the pHrodo LDL conjugate to serum-starved cells), you can clearly distinguish cell surface–bound vs. internalized pHrodo LDL complexes by measuring cell fluorescence using fluorescence microscopy, flow cytometry, or high-content analysis. The pHrodo LDL conjugates are multiplexable with other spectrally compatible fluorescent reagents.

 

Endocytosis of pHrodo Red LDL. HepG2 cells were plated onto poly-D-lysine–coated glass-bottom dishes, incubated in growth medium for 24 hr at 37°C, then switched to Gibco FluoroBrite DMEM with 0.3% BSA. After overnight incubation, cells were pretreated with vehicular control (0.3% BSA, left), 250 μg/mL LDL (center), or 250 μg/mL heparin (right) for 30 min. Invitrogen pHrodo Red LDL was added to the medium at 10 μg/mL, and cells were further incubated for 3 hr, counterstained with Invitrogen NucBlue Live ReadyProbes Reagent, washed twice, and imaged using the Invitrogen EVOS FL Auto 2 Imaging System. Images were matched for gain and exposure. In cells without pretreatment, the endocytosed pHrodo Red LDL complex (red) appears adjacent to the blue-fluorescent nuclei; in cells pretreated with unlabeled LDL or heparin, the pHrodo Red LDL remains outside of the cell and only dimly fluorescent.


Ready, set, flow! Ready Flow ready-to-use flow cytometry reagents

Invitrogen Ready Flow reagents are ready-to-use solutions designed to stain cells for analysis by flow cytometry without calculations, dilutions, or pipetting. These reagents simplify the most common flow cytometry assays that employ nucleic acid dyes, including dead-cell identification and cell cycle analysis, and are provided in convenient dropper bottles that can be stored at room temperature. Simply add 2 drops of Ready Flow reagent per 1 x 106 cells, incubate, and analyze.

Currently available Ready Flow reagents include a variety of fluorescent nucleic acid stains that are commonly used as cell viability and cell cycle reagents. With these Ready Flow reagents, preparing your samples for viability or cell cycle analysis by flow cytometry is quick and easy.

 

Cell cycle analysis with FxCycle Violet Ready Flow Reagent and the Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit. Jurkat cells (human T cell leukemia) were pulsed with 10 μM EdU for 2 hr prior to detection of proliferating cells with Alexa Fluor 647 azide using the Invitrogen Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit. Cells were then stained with 2 drops of Invitrogen FxCycle Violet Ready Flow Reagent and incubated for 30 min at 25°C. Data were acquired on an Invitrogen Attune NxT Flow Cytometer using a 405 nm laser and 440/50 nm emission filter. Analysis of the population indicates the following distribution: apoptotic sub-G1 cells, 3.4%; G0/G1, 49.1%; S, 33.0%; and G2/M, 14.0%.