This application protocol describes the flow cytometric analysis of major T cell subsets after spleen dissociation from healthy C57BL/6 mice. Viable single cells from mouse spleens are easily obtained using the gentleMACS™ Technology. For downstream flow cytometric analysis of T cells, we have designed a validated multicolor flow cytometry panel, using our REAfinity™ Recombinant Antibodies and Viobility™ Fixable Dyes. In addition, we provide an optimized gating strategy for the analysis of naive, central memory, and effector memory T cells, as well as the expression of CD127 and KLRG1 on CD4+ and CD8+ T cells.
PEB buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS® BSA Stock Solution (# 130-091-376) 1:20 with autoMACS®. Rinsing Solution (# 130-091-222). Keep buffer cold (2–8 °C). Always use freshly prepared buffer. Do not use autoMACS. Running Buffer or MACSQuant® Running Buffer as they contain a small amount of sodium azide that could affect the results.
Let the vials warm up to room temperature to avoid water condensation. Reconstitute one vial of lyophilized Viobility Fixable Dye by adding 100 μL anhydrous DMSO to the dye vial and mix until fully dissolved. Aliquot the solution and store at –20 °C under anhydrous conditions (desiccant) and protected from light for up to 1 month.
Configuration of the panel
|Viobility 405/520 Fixable Dye|
** is replaced by CD44 Antibody, anti-mouse, PE-Vio® 770, REAfinity™ (# 130-119-127)
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