Countess 1 fl automated cell counter

500 µL thawed PBMCs from each subject were added to 10 mL EasySep (StemCell Technologies, Cat. 20144) and centrifuged (300G, 5 min, room temperature). Extracellular nucleic acids were digested by resuspending cell pellets in 1 mL of buffer made from 18 mL EasySep and 21 µL Benzonase Nuclease (MilliporeSigma, Cat. 70664) and incubating (15 min, room temperature). Nuclease-treated cell-suspensions were then filtered through a 40 µm Flowmi Cell Strainer (Bel-Art, Cat. H13680-0040), centrifuged (300G, 5 min, room temperature), and finally resuspended in 100 µL EasySep buffer. Cell counting was performed on 1:100 dilutions of final cell suspensions stained with 0.4% trypan blue using a Countess I FL Automated Cell Counter (Thermo Fisher Scientific).

500 µL thawed PBMCs from each subject were added to 10 mL EasySep (StemCell Technologies, Cat. 20144) and centrifuged (300G, 5 min, room temperature). Extracellular nucleic acids were digested by resuspending cell pellets in 1 mL of buffer made from 18 mL EasySep and 21 µL Benzonase Nuclease (MilliporeSigma, Cat. 70664) and incubating (15 min, room temperature). Nuclease-treated cell-suspensions were then filtered through a 40 µm Flowmi Cell Strainer (Bel-Art, Cat. H13680-0040), centrifuged (300G, 5 min, room temperature), and finally resuspended in 100 µL EasySep buffer. Cell counting was performed using a Countess I FL Automated Cell Counter (Thermo Fisher Scientific) on 1:100 dilutions of final cell suspensions stained with 0.4% trypan blue.