Dutch modified rpmi 1640

Colonic biopsies were obtained at colonoscopy from three healthy female controls (ages 40, 57, and 66 years old) who had been referred for colorectal cancer screening and were macroscopically and histologically normal.
Paired samples were collected from the distal (left) and proximal (right) human colon (total of four biopsies from each area) in ice chilled PBS and processed within an hour. One biopsy from each compartment (proximal/distal) was used to assess the profile of intraepithelial lymphocytes (IEL) as described below while the other three biopsies were cultured in 1 ml of complete medium [Dutch modified RPMI 1640 (Sigma-Aldrich, Dorset, UK) containing 2 mM l-glutamine (Sigma-Aldrich) and 10% fetal calf serum (TCS cellworks, Buckingham, UK)] for 24 h in 12 well culture dishes and in the absence of antibiotics (1 biopsy/well) (37°C, 5% CO₂) in basal conditions or challenged with IL-15 (50 ng/ml, R&D) or LPS (Escherichia coli; 10 ng/ml Sigma). After 24 h, media from biopsy culture were centrifuged, filtered (0.2 μm), and cell/bacterial-free supernatants collected and preserved at −80°C. Negative controls included parallel processing (including 24 h incubation in culture dishes within the incubator and subsequent centrifugation and cryopreservation) of complete medium, which had not been conditioned with colonic samples.

Blood samples from the COVID-19 and post-COVID-19 patients were placed in heparin-lithium tubes and centrifuged over a Ficoll-Paque PLUS (Amersham Biosences, Chalfnt St Giles, UK) to obtain the plasma (which was immediately aliquoted at −80 °C until used) and PBMCs. The PBMCs were further washed in RPMI-1640 and cryopreserved at −80 °C in freezing medium (90% fetal calf serum and 10% DMSO) until needed. Plasma and PBMCs from the pre-pandemic controls were directly provided by the Spanish National Biobanks Network. Prior to use, the PBMCs were partially defrosted in a warm bath at 37 °C and subsequently washed in complete medium (Dutch modified RPMI 1640 (Sigma-Aldrich, Dorset, UK) containing 100 µg/mL penicillin/streptomycin, 2 mM L-glutamine, 50 µg/mL gentamicine (Sigma-Aldrich, Dorset, UK), and 10% fetal calf serum (TCS cellworks, Buckingham, UK)) before performing flow cytometry.

Blood obtained by venepuncture was diluted 1:1 (vol:vol) in PBS and layered over Ficoll-Paque Plus (Amersham Biosciences, Chalfont St. Giles, UK). After centrifugation at 800 g for 30 min at 18 °C, PBMC were collected at the interface. PBMC were resuspended in complete medium (Dutch modified RPMI 1640 (Sigma-Aldrich, Dorset, UK) containing 100 U/mL penicillin/streptomycin, 2 mM l-glutamine, 50 μg/mL gentamicin (Sigma-Aldrich) and 10% faetal calf serum (TCS cell works, Buckingham, UK)) for culture or in PBS for FACS analysis.