Thermo Fisher Scientific
LIVE/DEAD® Fixable Red Dead Cell Stain Kit
L34971
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We routinely perform FACS analysis of CD4+ and CD8+ T cells activated in vitro, assaying for transcription factor expression and proliferation. The in vitro activating stimulus that is used, i.e. anti-CD3/CD28, results in substantial cell death in the cultures, such that discrimination of live and dead cells is key to proper gating. Furthermore, because many of the analyses require staining for transcription factors, a fixable dye is necessary. We have used several different colors from the Live/dead dye series by Thermo Fisher. The Red variant is particularly useful for cultures with GFP-reporter T cells. The Live/dead red dye is critical for many of our in vitro experiments.
Flow cytometry
Mouse CD4+ T cells stimulated for 3 days in vitro
CD4+ T cells were harvested from in vitro stimulation cultures and washed with dPBS. Cells were then resuspended in Live/Dead Red dye diluted at 1:3000 in dPBS, incubated for 20 minutes on ice, and then washed once more with excess dPBS.
Requires ArC™ Amine Reactive Compensation Beads for proper compensation
The Live/Dead Red dye provided efficient separation of dead cells within the lymphocyte gate of our flow cytometry analyses of T cells. The dye was extremely potent and could be used at dilutions on the order of 1:3000 without altering the voltages on our BD Fortessa. No compensation issues were observed with other channels on the yellow laser, e.g. PE or PE-Cy7, and the dye was easily integrated into multi-color panels. Fluorescence was well-maintained with aldehyde fixation. Staining required only a 15-20 minute incubation.
N/A
Extremely bright signal and excellent separation
None
Excellent fixable dye for separation of live and dead cells that can be used at very dilute concentrations.