PE Annexin V Apoptosis Detection Kit I

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Treated with FITC Annexin V and PI and incubated for 15 min at RT (25°C) in the dark

Protocol tips
- Treated with FITC Annexin V and PI and incubated for 15 min at RT (25°C) in the dark

Publication protocol

Spleens and lymph nodes were harvested on days +31, 33 or 35 post-BMT and flow cytometric-based enumeration of lymphocytes was accomplished using a FACS Calibur equipped with CellQuest software version 5.2.1 (Becton Dickinson, San Jose, CA). Briefly, 1 × 106 freshly isolated, erythrocyte-depleted splenocytes or lymph node cells were treated with anti-FcγIII/II receptors monoclonal antibody (clone 2.4G2) and then stained at 4°C for 20 minutes with a monoclonal antibody cocktail containing Vβ6 (for Marilyn) or Vβ8.3 (for Matahari)-PE, CD45.2-PerCP Cy5.5 and CD45.1-APC (BD Pharmingen, San Jose, CA), then washed in FACS buffer (Phosphate buffered salt solution with 0.2% fetal calf serum and 0.1% sodium azide). To examine for apoptosis, Annexin-V-PE staining was performed according to the manufacturer's instructions (Becton Dickinson, San Jose, CA). For phenotypic analysis of Marilyn and Matahari T cells in vivo, the following antibodies were used: anti-CD107a-PE (clone 1D4B, BD Pharmingen), anti-PD1-APC (clone J43, eBiosciences), and anti-Tim3 (clone RMT3-23, eBiosciences). For intracellular staining of interferon gamma (IFNγ), cells were fixed and permeabilized according to the manufacturer's instructions using Fix and Perm buffer (BD Pharmingen) and stained with anti-IFNγ-PE (clone XMG1.2, eBiosciences).

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Manufacturer protocol

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