This application protocol describes the flow cytometric analysis of macrophages 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 macrophages, 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 macrophages from mouse spleen.
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|
|CD335 (NKp46)*||PE-Vio 615||REA815|
|* Exclusion channel containing lineage markers to exclude T cells (CD3ε), B cells (CD19), and NK cells (CD335 (NKp46)) from the analysis.|
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