PMC:7402624 / 64080-65577 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7402624","sourcedb":"PMC","sourceid":"7402624","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7402624","text":"Antibody panels and staining\nApproximately 1-5×106 freshly isolated PBMCs were used per patient per stain. See table S7 for buffer information and table S8 for antibody panel information. PBMCs were stained with live/dead mix (100 μl, 10 min, RT), washed with FACS buffer, and spun down (1500 rpm, 5 min, RT). PBMCs were incubated with 100 μl of Fc block (RT, 10 min) before a second wash (FACS buffer, 1500 rpm, 5 min, RT). Pellet was resuspended in 25 μl of chemokine receptor staining mix, and incubated at 37°C for 20 min. Following incubation, 25 μl of surface receptor staining mix was directly added and the PBMCs were incubated at RT for a further 45 min. PBMCs were washed (FACS buffer, 1500 rpm, 5 min, RT) and stained with 50 μl of secondary antibody mix for 20 min at RT, then washed again (FACS buffer, 1500 rpm, 5 min, RT). Samples were fixed and permeabilized by incubating in 100 μl of Fix/Perm buffer (RT, 30 min) and washed in Perm Buffer (1800 rpm, 5 min, RT). PBMCs were stained with 50μl of intracellular mix overnight at 4°C. The following morning, samples were washed (Perm Buffer, 1800 rpm, 5 min, RT) and further fixed in 50 μl of 4% PFA. Prior to acquisition, samples were diluted to 1% PFA and 10,000 counting beads added per sample (BD, Cat#335925). Live/dead mix was prepared in PBS. For the surface receptor and chemokine staining mix, antibodies were diluted in FACS buffer with 50% BD Brilliant Buffer (BD, Cat#566349). Intracellular mix was diluted in Perm 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