LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit, for UV excitation

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 3 × 10^6	Add reconstituted fluorescent reactive dye and incubate at room temperature or on ice for 30 minutes

Upstream tips
Seed 3 × 10^6
Protocol tips
Add reconstituted fluorescent reactive dye and incubate at room temperature or on ice for 30 minutes

Publication protocol

To detect intracellular IL‐10, spleen cells (3 × 106) were restimulated for 5 h with PMA/ionomycin in the presence of Monensin and Brefeldin A (Biolegend). Cells were stained with Live/Dead Fixable Blue Dead Cell Stain Kit (Life Technologies), Zombie UV™ Fixable Viability Kit (Biolegend) or Fixable Viability Dye eFluor® 450 (Affymetrix eBioscience) according to the manufacturers’ instructions. For surface staining, cells were stained with anti‐mouse CD4‐Allophycocyanin‐, ‐Phycoerythrin‐Cy7 (PE‐Cy7), ‐Brilliant Violet (BV) 421, ‐BV510 (clone: RM4‐5), anti‐mouse CD8‐Allophycocyanin (clone: 53–6.7), anti‐mouse CD272 (BTLA)‐PE (clone: 6F7), anti‐mouse CD279 (PD‐1)‐Allophycocyanin or ‐Biotin (clone: RMP1‐30), anti‐mouse CD19‐BV510 or ‐Allophycocyanin (clone: 1D3) and Streptavidin BV421. Antibodies were used in 1:200 dilutions for 30 min on ice. For intracellular expression of CTLA‐4, IL‐10, and Foxp3, cells were stained with anti‐mouse CTLA‐4‐BV421 or ‐Allophycocyanin (clone: UC10‐4B9), anti‐mouse IL‐10 PE (clone: JES5‐16E3), and anti‐mouse/anti‐rat Foxp3‐Alexa Fluor 700 (AF700), ‐PE or ‐Allophycocyanin Staining Set (FJK‐16s) using Foxp3 Staining Set (eBioscience) according to the manufacturer's instructions. For analysis of reconstitution of RAG‐1−/− mice, spleen cells were stained with anti‐mouse CD4, anti‐mouse CD8, and anti‐mouse CD19 Ab. Ab were purchased from BioLegend or Affymetrix eBioscience. Samples were analyzed on a LSRII Flow Cytometer (Becton Dickinson) using FlowJo software (TreeStar).

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Manufacturer protocol

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