Versene solution

Flavin mononucleotide (riboflavin-5′-phosphate) was obtained from Pharmstandard (Russia). Phosphate buffered saline (PBS, pH 7.4) was purchased from Lonza (Switzerland). RPMI-1640 cell growth medium, L-glutamine, and Penicillin-Streptomycin (Pen Strep, 100x) were purchased from Gibco (UK), fetal bovine serum (FBS) was from HyClone (USA). MTT dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), dimethyl sulfoxide (DMSO), Versene solution, and paraformaldehyde were from Sigma (USA). CellROX Deep Red fluorescent dye, Annexin V-FITC Apoptosis Detection Kit, Hematoxylin and Eosin were from Thermo Fisher Scientific (USA). BD Matrigel™ Basement Membrane Matrix was from BD Biosciences (USA).

Ethical approval for these studies was provided by the National Research Ethics Service Committee South Central—Southampton A, ref. 13/SC/0043. Peripheral blood mononuclear cells (PBMCs) were isolated from single-donor buffy coats from the National Health Service Blood and Transplant, Southampton, United Kingdom. Leukocytes were isolated by density gradient centrifugation over Ficoll-Paque (GE Healthcare Life Sciences, United Kingdom). Isolated PBMCs were infected with M. tuberculosis Lux at a multiplicity of infection (MOI) of 0.1 and kept overnight at 37°C in a 5% CO₂ incubator in RPMI 1640 medium supplemented with 10 μg/ml ampicillin, 2 mM glutamine, 25 µg/ml kanamycin, and 10% fetal bovine serum (Labtech International Ltd.). The next day, infected PBMCs were transferred from vented flasks to 50-ml Falcon tubes after detachment with Versene solution (Sigma) for 10 min and scraping. After Hanks balanced salt solution (HBSS) without Ca/Mg (Gibco) was added, cells were centrifuged at 320 × g for 8 min at 4°C and the supernatant was decanted. The pelleted cells were resuspended in appropriate volumes of RPMI 1640 medium supplemented with 10 μg/ml ampicillin, 2 mM glutamine, 25 µg/ml kanamycin, and 10% human AB serum (Sigma), referred to as complete RPMI medium.

Monocytes were isolated using negative MACS isolation with the Pan monocyte isolation kit (Miltenyi Biotech). Briefly, stimulated PBMCs were washed with PBS and incubated with versene solution (0.48mM EDTA, Sigma Aldrich) for 30 minutes at 37°C. Cells were scraped from the plates, counted, spun down and resuspended in MACS isolation buffer (PBS with 0.5% BSA and 2mM EDTA).
Monocytes from COVID-19 patients were isolated directly after isolation of PBMCs. PBMCs were counted, spun down and resuspended in MACS isolation buffer. Monocyte isolation was performed according to manufacturer’s instructions.