Alexafluor af 488 nhs ester

Cells were live/dead stained using the Live/Dead Fixable Near IR Dead
Cell Stain Kit (Life Technologies). For surface stains, cells were stained for
30 minutes on ice with indicated antibodies. Clones, sources & dilutions
can be found in the Supplementary Table 5. For intracellular stains, samples were fixed
at the indicated time after stimulation in 2% paraformaldehyde, surface
stained with CD25-biotin and CD4-BUV395, fixed again and permeabilized with ice
cold 90% methanol at −20 °C overnight. Samples were then
barcoded using Pacific Orange-NHS ester (0.33 or 5 μg/mL), Pacific
Blue-NHS Ester (0.67 or 10 μg/mL), and AlexaFluor (AF) 488-NHS Ester
(0.26 or 2 μg/mL) (Life Technologies), as previously described³⁶ . Intracellular antigens were
then stained for 30 minutes at 23 °C with antibodies indicated in Supplementary Table 5.
Samples were acquired on a BD LSR Fortessa and analyzed in FlowJo (Tree
Star).

Cells were live/dead-stained using the Live/Dead Fixable Near IR Dead Cell Stain Kit or Live/Dead Fixable Violet Dead Cell Stain Kit (Life Technologies). For surface stains, cells were stained for 30 minutes on ice with the indicated antibodies. Information on antibody clones, sources, and working dilutions are listed in Table S1. For intracellular stains, samples were fixed at the indicated time after stimulation in 2% paraformaldehyde, surface stained with CD8a-BUV737 and CD4-BUV395, fixed again, then permeabilized with ice cold 90% methanol at −20 °C overnight. Samples were then barcoded using Pacific Orange-NHS ester (0.33 or 5 μg/mL), Pacific Blue-NHS Ester (0.67 or 10 μg/mL), and AlexaFluor (AF) 488-NHS Ester (0.26 or 2 μg/mL) (Life Technologies), as previously described(5 (link)). Intracellular antigens were then stained for 30 minutes at 23 °C with antibodies indicated in Table S1. Samples were acquired on a BD LSR Fortessa SORP and analyzed in FlowJo (Tree Star).