Tim 3 pacific blue

Flow cytometry antibodies used for surface marker staining are listed as follows. Antibodies from BD Biosciences include: CD3‐BUV395 (Clone UCHT1; cat #563 546), CD4‐BV786 (Clone SK3; cat #563 877) and CD8‐PE‐Cy7 (Clone SK1; cat #335 787). Antibodies from Biolegend include: CD3‐FITC (Clone UCHT1; cat #300 406), CCR7‐PE (Clone G043H7; cat #353 204), CD45RA‐APC (Clone HI100; cat #304 112), CD45RO‐Pacific Blue (Clone UCHL1; cat #304 215),CD27‐Pacific Blue (Clone M‐T271; cat #356 413), CD28‐FITC (Clone CD28.2; cat #302 906), CD28‐PE (Clone CD28.2; cat #302 907), CD95‐FITC (Clone DX2; cat #305 605), CD127‐Brilliant Violet 785 (Clone A019D5; cat #351 329), TIM‐3‐Pacific Blue (Clone F38‐2E2; cat #345 041), CD57‐FITC (Clone HNK‐1; cat #359 603), LAG‐3‐Brilliant Violet 785 (Clone 11C3C65; cat #369 321) and human TruStain FcX reagent (Cat #422 302). Antibodies for intracellular staining include granzyme B‐PE (BD Biosciences, GB11; cat #561 142), IFN‐γ‐FITC (Miltenyi Biotec, cat #130‐090‐433), IL‐2‐PE (Miltenyi Biotec, cat #130‐090‐487) and TNF‐α‐APC (Miltenyi Biotec, cat #130‐091‐267).

After 2–4 days, B cells were gently harvested and incubated with fluorochrome-labeled detection antibodies for 30 min at 4 °C in the dark, washed two times, and resuspended in 400 µL washing buffer (PBS containing 0.05% bovine serum albumin). Fluorescence was detected with a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA) and analyzed with Kaluza software from Beckman Coulter. The following antibodies were used for detection: BTLA Brilliant Violet 421 (344512), CD19 APC-Fire 750 (302257), GITR Brilliant Violet 421 (371207), OX40 PE-Cy5 (350009) and TIM-3 Pacific Blue all from Biolegend (San Diego, CA, US), CD137 PE (12-1379-42), CD27 PE-Cy5 (15-0279-42), CD39 PE-Cy7 (25-0399-42), CD73 FITC (11-0739-42), LAG3 PE (12-2239-42) and PD-1 PE (12-2799-41) from eBioscience (San Diego, CA, US), CD86 Alexa Fluor 700 (561124) and CTLA4 PE-Cy5 (561717) from BD Biosciences (Franklin Lakes, NJ, USA).