Pacific orange nhs ester

The following FCB dyes were used: CBD500 (BD Biosciences, San Jose, CA, USA); Pacific Orange NHS ester, DyLight 350 NHS ester, and DyLight 800 NHS ester (Thermo Fisher Scientific, Waltham, MA, USA). Antibodies tested for surface staining were: CD3-BV605 (OKT3) (BioLegend, San Diego, CA); CD4-APC (RPA-T4) (BD Biosciences, San Jose, CA, USA); CD8-PE-Cy5 (B9.11), and tube B of IOTest Beta Mark, containing Vβ 9-PE, Vβ 17-PE/FITC, and Vβ 16-FITC (FIN9, E17.5F3, and TAMAYA1.2) (Beckman Coulter, Miami, FL). LIVE/DEAD Fixable Aqua (a viability dye) for 405 nm excitation was used to exclude dead cells from analysis (Thermo Fisher Scientific). Aqua dye was dissolved in DMSO and stored at −80°C, according to the manufacturer’s instructions. Just before use, Aqua dye was diluted 1:16 with PBS and used for staining. All buffers (Phosflow Lyse/Fix Buffer 5X, Phosflow Perm Buffer II, and Phosflow Barcoding Wash Buffer 4X; BD Biosciences) were prepared, according to the manufacturer’s instructions.

The following FCB dyes were used: DyLight 350 NHS ester and Pacific Orange NHS ester (Thermo Fisher Scientific, Waltham, MA, USA). Antibodies used for surface staining were: mouse anti-human CD3-PerCP-Cy5.5 (clone SK7), mouse anti-human CD4-PE-Cy7 (clone SK3), mouse anti-human CD8-FITC (clone RPA-T8), and mouse anti-human CD20-APC-H7 (clone H1) (BD Biosciences, San Jose, CA, USA); and CD14-PE (clone M5E2) from BioLegend (San Diego, CA). Antibodies used for phosphoproteins were: pSTAT1(pY701)-Alexa Fluor 647 (clone 4a), pSTAT3(pY705)- Alexa Fluor 647 (clone 4/P-STAT3), and pSTAT5(pY694)- Alexa Fluor 647 (clone 47/Stat5 pY694) (BD Biosciences). Phosflow Lyse/Fix Buffer 5X, Phosflow Perm Buffer III, and Phosflow Barcoding Wash Buffer 4X buffers (BD Biosciences) were prepared and used according to manufacturer’s instructions. Phosflow Perm Buffer II (BD Biosciences) was diluted 1:1 with cold PBS and kept on ice before use. For PBMC stimulation, the following cytokines were used: recombinant human IL-10 (PeproTech, Rocky Hill, NJ); human Interferon-α (Cell Signaling Technology, Boston, MA); recombinant human IL-2 (Hoffmann-La Roche Inc, Nutley, NJ). The CTL Anti-Aggregate (CTL) wash buffer was from Cellular Technology Limited (Shaker Heights, OH).

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).