Fixable Viability Dyes
Determining cell viability is crucial when assessing a cells response to treatment in order to exclude them from final data analysis. When performing intracellular immunophenotyping by flow cytometry, a fixable viability dye is critical to preserve the staining pattern after fixation, in order to properly identify cell populations. Numerous fixable viability stains have been created to quantify viability, however they are not compatible with fixation protocols, and ultimately may result in artifacts being generated within the data set.
The iQue® Fixable Viability Dyes provide accurate assessment of cell viability in samples after fixation and/or permeabilization in a 96-well format with a streamlined workflow from cell labeling to analysis. Obtaining accurate cell viability for intracellular flow cytometry workflows has never been easier!
Application
Assay Concept
iQue® Fixable Viability Dyes are amine-reactive membrane impermeable dyes that discriminate between live and dead cells. The stained cells can be fixed and permeabilized, making the dyes compatible with downstream intracellular staining without any loss of intensity.
The iQue® Fixable Viability Kits have been validated to run on the iQue® platform with BR and VBR configurations. It enables quantitative analysis of cell viability, cell surface and intracellular targets with a streamlined workflow from cell labeling to analysis. iQue® Fixable Viability Kits are available in three different colors for multi-color flow panel needs. Pre-set templates are included in the kit for gating strategy and analysis for the ease of use.
Basic workflow:
- Wash cells with protein-free PBS
- Stain cells with dye diluted in protein-free PBS for 20-30 minutes
- Perform downstream staining protocols (surface marker staining, fixation/permeabilization, intracellular staining) if needed.
- Analyze the plate on iQue® platform (BR and VBR configurations).
Key Advantages
Preserve staining post fixation - Maintain fluorescence staining intensity to discriminate live and dead cells post fixation or permeabilization.
Enable artifact-free analysis of intracellular targets - Monitor surface and intracellular marker expression simultaneously while reducing artifacts from dead cells.
Save precious samples - Conserve samples for use in multiple downstream assays with only 20 µL of cells required.
Preserve staining post fixation
Retain fluorescence performance to discriminate live and dead cells without any loss of staining intensity post fixation or permeabilization.
Figure 1. Comparison of fluorescent staining with iQue® Fixable Viability Dyes in fixed/permeabilized cells and non-fixed cells.
A mixture of viable and heat-treated (65°C, 10 minutes) non-viable Jurkat cells were stained with iQue® Fixable V/Blue (A), B/Green (B), or R/Red (C) dye. The stained cells were either unfixed or fixed and permeabilized with 3.7% formaldehyde, 0.1% saponin in PBS. All the samples were acquired with an iQue® instrument and histograms were derived from gated singlets using the pre-set templates included in the kit.
Enable artifact-free analysis of intracellular targets
Analyze surface and intracellular marker expression simultaneously while minimizing the potential artifacts from dead cells.
Figure 2. Example of Detection and Analysis of intracellular IFN-γ expression in iQue® Fixable Viability Dye-stained PBMCs.
PBMCs were cultured in CD3 antibody-coated plate for two days in the presence of 10 ng/mL human recombinant IL-2. Protein transport inhibitor monensin (2 µM) was added to the cell culture 4 hours before the assay. Activated PBMCs (20 µL) were added to each well of a 96-well assay plate, stained with iQue® Fixable Viability (V/Blue) Dye, followed by staining with T cell surface marker CD3. The cells were then fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences), stained with PE-conjugated mouse anti-human IFN-γ antibody. The samples were acquired with an iQue® instrument and data were analyzed with Forecyt software. A, Gate of all cells. B, Gate of singlets. C, Exclusion of dead cells with iQue Fixable Viability (V/Blue) Dye staining. D, Plot of IFN-γ expression in CD3+ live cells.
Save precious samples
Conserve samples to accommodate multiple down stream assays with only 20 µL of cells required.
Figure 3. Easy to follow, low volume (20 µL/well) protocol save cells and setup time
Ordering Information
Platform: Compatible with iQue® 3/iQue® Screener Plus - VBR and BR Configuration | |||
| Product | Size | Catalog Number | |
| iQue® Fixable Viability (V/Blue) Kit | 5 x 96 wells | ||
| iQue® Fixable Viability (B/Green) Kit | 5 x 96 wells | ||
| iQue® Fixable Viability (R/Red) Kit | 5 x 96 wells | ||
