LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Add 1 μL of the reconstituted fluorescent reactive dye. Incubate at room temperature or on ice for 30 minutes, protected from light.	Detect at ~525 nm and use filters 525/50 nm or similar.

Protocol tips
Add 1 μL of the reconstituted fluorescent reactive dye. Incubate at room temperature or on ice for 30 minutes, protected from light.
Downstream tips
Detect at ~525 nm and use filters 525/50 nm or similar.

Publication protocol

For the analysis of CD62L and CD44 expression, enriched γδ T cells were stained with LIVE/DEAD Aqua, Pacific blue CD62L (MEL-14), FITC-CD44 (IM7), and PE. For the analysis of Thy1.1 expression on cells isolated from IL-17fThy1.1/Thy1.1 reporter mice, enriched γδ T cells were stained with LIVE/DEAD Aqua, Pacific blue-CD62L, FITC-Thy1.1 (OX-7; BioLegend), PE, APC-GL-3. For the analysis of IL-1R (CD121a) and CD27, enriched γδ T cells were stained with LIVE/DEAD Aqua, Pacific blue-CD62L, FITC-CD27 (LG.7F9), PE, APC-CD121a (JAMA-147; BioLegend), PerCP Cy5.5-GL-3, eFluor-605NC-CD44. All staining include the addition of APC-Cy7 conjugated anti-TCRβ, B220, CD11b, CD11c, Gr-1, F4/80. APC-Cy7 and Aqua positive cells were excluded from analysis.

For intracellular IL-17 staining, γδ T cells were enriched from pooled draining lymph node cells from 5-15 of immunized mice. Enriched γδ T cells were re-stimulated in vitro with PE (0.5 μM), IL-1 and IL-23 (1ng/ml each) for seven hours before analysis. Cells were then harvested, blocked with serum, FcBlocker and dump antibodies as described above, and fixed and permeabilized with BD Cytofix/Cytoperm solution (BD Biosciences) for 20 min on ice, followed by staining with APC conjugated IL-17A (eBio17B7) or Rat IgG2a isotype. Analysis of Vγ usage was as described (Shin, et al.,2005).

Human and bovine PBL γδ T cells were stained with PE (0.5 μM, P1 strain), without enrichment. Human cells were also stained APC conjugated anti-γδ TCR (5A6.E9; Invitrogen Molecular probes), Live/Dead Aqua, FITC conjugated anti-CD19 (HIB19), αβ TCR (IP-26), CD14 (HCD14), CD16 (3G8). Bovine cells were stained with APC conjugated anti-γδ TCR (GB21A) (all antibodies are from VMRD Inc.), LIVE/DEAD Aqua, FITC conjugated anti-CD4 (CC8), CD8 (CC63), CD14 (CC-G33; all from AbD Serotec), anti-B lymphocytes (LCT30A). Aqua and FITC positive cells were excluded from analysis.

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Manufacturer protocol

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