Anti cd19 phycoerythrin pe cy7

To isolate cells from inflamed tissues, lung tissues were cut into four pieces and gently stirred in flasks with solution [PBS containing 25 mL 10 mmol/L EDTA, 3% fetal bovine serum (HyClone Laboratories, Logan, UT), 20 mmol/L HEPES, and 1 mmol/L sodium pyruvate) for 30 minutes at 37°C. The segments were washed three times with PBS and digested with 5 mL RPMI 1640 medium containing 1 mg/mL of type V collagenase (Sigma-Aldrich) for 45 minutes at 37°C. Finally, the soup containing ear total cell was centrifuged and cultured in T-cell media. For the surface marker staining, cells were washed with ice-cold PBS, resuspended in 100 μL of PBS, and stained with anti–CD45-APC-R700 (BD Biosciences, Franklin Lakes, NJ), anti–T-cell receptor beta (TCRb)-APC/Fire750 (BD Biosciences), anti–MHCII-peridinin chlorophyll protein complex (PerCP)-Cy5.5 (BD Biosciences), and anti–CD19-phycoerythrin (PE)/Cy7 (Biolegend, San Diego, CA). Dead cells were excluded using LiveDead Fixable Viability dye (Invitrogen). Samples were acquired using ID7000 (Sony, Tokyo, Japan), and data were analyzed using FlowJo software version 10 (Tree Star, Ashland, OR).