Flowmi 40 m cell strainers

BAL fluid: Approximately 10 ml of BALF was obtained and placed on ice, with processing within 3 h in a BSL-3 laboratory. BAL fluid was centrifuged and the supernatant was frozen at −80 °C for further experiments. The cellular fraction was resuspended in ice-cold PBS and samples were filtered using 40 µm nylon mesh (ThermoFisher Scientific). Following centrifugation, the supernatant was decanted and discarded, and the cell pellet was resuspended in red blood cell lysis buffer. Following a 5-min incubation at room temperature, samples were centrifuged and resuspended in PBS containing UltraPure BSA (AM2616, ThermoFisher Scientific) and filtered over Flowmi 40 µm cell strainers (VWR) using wide-bore 1 ml low-retention filter tips (Mettler-Toledo). Next, 10 µl of this cell suspension was counted using an automated cell counter to determine the concentration of live cells. The entire procedure was completed in <1.5 h.
PBMCs: PBMC samples were thawed, centrifuged and the resulting cellular fraction resuspended in PBS containing UltraPure BSA. This was followed by filtering and counting, according to BALF protocol. The entire procedure was completed in <1 h.

Fresh primary lesions of 11 patients were isolated immediately following tumor resection and transferred to a 50 mL centrifuge tube filled with precooled RPMI 1640 medium with 0.04 % bovine serum albumin (BSA, Gibco, Carlsbad, CA, USA). They were then quickly transported on ice to the FDZSH laboratory to minimize the ischemic time. Samples were cut into 1 mm3 pieces, followed by enzymatic digestion using the Miltenyi Tumor Dissociation Kit (Miltenyi, Bergisch Gladbach, Germany). The samples were then centrifuged at 300 g for 30 s, and the supernatant was discarded. Next, 1× PBS (calcium and magnesium free) containing 0.04 % BSA (400 µg/ml) was added and then centrifugation at 300 g for 5 min. The cell pellet was resuspended in 1 ml red blood cell lysis buffer and incubated for 10 min at 4 ℃. The samples were then resuspended in 1 ml PBS containing 0.04 % BSA. The samples were then filtered using Scienceware Flowmi 40-µm cell strainers (VWR). Finally, 10 µL of suspension was counted under an inverted microscope with a hemocytometer. Trypan blue was used to quantify the live cells.