eFluor® Fixable Viability Dyes
These fluorescent dyes bind free amine groups on both surface and intracellular proteins. Live cells are not membrane permeable, therefore the dyes only minimally label live cells; dead cells with compromised membranes readily take up the dyes, resulting in a brightly fluorescence dead cell population. Unlike 7-AAD and Propodium Iodide, Fixable Viability eFluor® Dyes are retained by dead cells throughout fixation, permeabilization and cryopreservation.
Excluding dead cells from analysis is a recommended procedure for all intracellular staining protocols including detection of nuclear factors, transcription factors, cytosolic signaling molecules and secreted growth factors and cytokines.
- Retained in fixed, cryopreserved samples
- Suitable for cells from all species
- Available dye options for both the red and violet laser
- Tight emission spectra allows easy integration into multicolor experiments
Eliminate dead cells from analysis. Obtain clear and accurate identification of rare cell populations, such as regulatory T cells (Treg)
Mouse splenocytes were stimulated with PMA and Ionomycin for 12 hours, then stained with Fixable Viability Dye eFluor 780. Cells were surface stained with Anti-Mouse CD4 PerCP-Cy5.5, followed by intracellular staining with Anti-Mouse/Rat Foxp3 Alexa Fluor 488 using the Foxp3 Concentrate and Diluent buffers. The top data plots describe the analysis of CD4 and Foxp3 staining when the gating strategy is based solely on forward scatter and side scatter. Results show poor separation of Foxp3+ cells. The bottom row show the analysis of CD4 and Foxp3 staining using a live gating strategy with Fixable Viability Dye eFluor 780. Analysis reveals a discrete population of CD4+Foxp3+, indicative of a Treg cell population.
Eliminate false positives with live cell gating strategy.
Mouse splenocytes were cultured overnight and then stained with Fixable Viability Dye eFluor 506, followed by surface staining with Anti-Mouse CD19 APC. The top row shows analysis of CD19 staining when gating strategy is based on forward scatter and side scatter. The bottom row shows analysis of CD19 staining when employing a live cell gating strategy based on Fixable Viability Dye eFluor 506. A shoulder of CD19+ cells appearing in the top right histogram is due to dead cells non-specifically staining CD19+. Staining is completely eliminated after gating on the live cells, as shown in the bottom right histogram.
Propidium Iodide (PI) and 7-AAD Viability Dyes
PI and 7-AAD dyes are ready-to-use solutions for the exclusion of nonviable cells in flow cytometric analysis. PI binds to double stranded DNA, but is excluded from cells with intact plasma membranes. PI can be detected with the PerCP tandem dye channel for viability exclusion, but should be analyzed in the PE channel when used as a counterstain for FITC Annexin V. 7-AAD can be used in place of PI. The advantage of 7-AAD over PI is that there is minimal spectral overlap between these emissions. Fluorescence is detected in the far red range of the spectrum (650 nm longpass filter). PI and 7-AAD can also be used for flow cytometric analysis of the cell cycle.