Anti human cd3 ucht1 pe cy7

PBMCs were resuspended at 1 × 10⁶ cells/ml in FACS buffer with 20% human Fc receptor–binding inhibitor (eBioscience) and incubated for 20 min at room temperature in 5-ml polystyrene FACS tubes (BD Biosciences). The PBMCs were stained in a total volume of 135 μl per tube with 0.5 μl of anti-human CD3 (UCHT1) PE-Cy7 (eBioscience), 0.5 μl of anti-human CD4 (SK3) PerCP-eFluor 710 (eBioscience), 0.5 μl of anti-human CD8 (SK1) APC-eFluor780 (eBioscience), and anti-human CD14 (MφP9) V500 (BD Biosciences). The cells were incubated in the dark for 30 min at room temperature. They were washed twice with 3 ml of FACS buffer and resuspended in 0.5 ml of FACS buffer with 1 μM 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) (fig. S11).

PBMC suspensions were acquired using an eight-color FACSVerse flow cytometer (BD Biosciences) with 405-, 488-, and 640-nm laser excitations at an average flow rate of 2 μl/s and an average threshold event rate of 1000 to 2000 events/s. Multicolor Cytometer Setup and Tracking beads (BD Biosciences) were used for QC and standardization of photomultiplier tube detector voltages across multiple experimental runs. Fluorescence compensation for immunophenotyping was conducted using anti-mouse immunoglobulin G (IgG)κ antibody capture beads (Bangs Laboratories) labeled separately with each antibody. Fluorescence compensation for intracellular staining of cell signaling epitopes was conducted using anti-mouse IgGκ antibody capture beads labeled separately with anti-human CD3 (UCHT1) PE-Cy7 (eBioscience), anti-human Stat3 (pY705) (4/P-STAT3) Alexa Fluor 488 (AF 488; BD Biosciences), anti-human Stat3 (pY705) (4/P-STAT3) PE (BD Biosciences), and anti-human Stat3 (pY705) (4/P-STAT3) AF 647 (BD Biosciences; KP and TI) or anti-human CD3 (UCHT1) APC (eBioscience) and PLC-γ1 (10/PLCgamma) PE (BD Biosciences; DR and CV), alongside single-stain controls with maximum and minimum concentrations of each barcoding dye per PBMC sample.