Alexa Fluor® 488 Rat Anti-Mouse CD146

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Spleens, lymph nodes, lungs, and livers were harvested for IL-17 analysis on d28, d42, and d56, and single cell suspensions were made. The cells were stimulated with Cell Stimulation Cocktail plus Protein Transport Inhibitors (eBioscience, 00-4975-93) for 5 hours at 37°C in complete-RPMI.	Cells were stained with fixable viability dye eF780 or eF506 (eBioscience). All cells were fixed and permeabilized by the FOXP3 Fixation/Permeabilization Concentrate and Diluent kit (eBioscience).

Upstream tips
Spleens, lymph nodes, lungs, and livers were harvested for IL-17 analysis on d28, d42, and d56, and single cell suspensions were made. The cells were stimulated with Cell Stimulation Cocktail plus Protein Transport Inhibitors (eBioscience, 00-4975-93) for 5 hours at 37°C in complete-RPMI.
Protocol tips
Cells were stained with fixable viability dye eF780 or eF506 (eBioscience). All cells were fixed and permeabilized by the FOXP3 Fixation/Permeabilization Concentrate and Diluent kit (eBioscience).

Publication protocol

Spleens, lymph nodes, lungs, and livers were harvested for IL-17 analysis on d28, d42, and d56, and single cell suspensions were made. The cells were stimulated with Cell Stimulation Cocktail plus Protein Transport Inhibitors (eBioscience, 00-4975-93) for 5 hours at 37°C in complete-RPMI. Cells were stained with fixable viability dye eF780 or eF506 (eBioscience), anti-CD4 (eBioscience, clone GK1.5), anti–IFN-γ (BD Biosciences, clone XMG1.2), anti–IL-17A (eBioscience, clone eBio17B7), anti–IL-21 (eBioscience, clone FFA21), anti-RORγt (eBioscience, clone B2D), anti-CCR5 (eBioscience, clone HM-CCR5 [7A4]), anti-CD146 (BD Biosciences, clone ME-9F1), and anti-CD45.2 (BD Biosciences, clone 104). All cells were fixed and permeabilized by the FOXP3 Fixation/Permeabilization Concentrate and Diluent kit (eBioscience).

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