Red blood cell lysis buffer

In some experiments, blood was collected from the tail vein into EDTA-coated Eppendorf tubes and further treated with red blood cell lysis buffer (Sigma–Aldrich) prior to Ab staining. Lungs and female genital tracts (GTs) were harvested from perfused female mice and broncho-alveolar lavage (BAL) fluid was also collected where indicated as described previously [19] (link).
Single cell suspensions from the spleen were prepared in complete RPMI medium (RPMI 1640) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine (Invitrogen), 50 μM 2-mercaptoethanol (Invitrogen) and 10% heat inactivated FBS (Biosera). Erythrocytes were depleted by treatment with red blood cell lysis buffer (Sigma–Aldrich). Single cell suspensions from the mucosal tissues were obtained by enzymatic digestion for 30 min at 37 °C in complete RPMI 1640 containing 1 mg/ml Collagenase D (RocheTM) and 0.02 mg/ml DNase I from bovine pancreas (RocheTM).

Single cell suspensions were made from murine mesenteric lymph nodes (MLN) by maceration through 70 μm filters (BD) into complete RPMI 1640 (cRPMI) medium containing HEPES (Gibco), supplemented with 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco), 10% heat-inactivated foetal calf serum (Gibco). Contaminating red blood cells were removed by resuspending the cells from one spleen in 2 ml of red blood cell lysis buffer (Sigma) and incubating at RT for 2 min. Cells were then washed with cRPMI and counted on a haemocytometer by trypan blue exclusion.
For flow cytometry analysis of murine blood taken at 21 days post naïve T cell transfer, a few drops of blood were collected into several millilitres of FACS buffer containing EDTA (to prevent coagulation), centrifuged for 5 min at 1500 g and pellets resuspended in 3 ml of red blood cell lysis buffer (Sigma), incubated for 5 min at room temperature, topped up with FACS buffer and recentrifuged for 5 min at 1500 g. Cells were then used for subsequent flow cytometry staining and analysis (see below).

Biopsies were minced into small pieces with a scalpel and incubated at 37 °C for 5 minutes in freshly prepared dissociation buffer containing 2 mg/mL Collagenase P (Roche) and 0.2 mg/ml DNase I (Roche). Dissociated tissue was harvested and filtered through a 40 µm cell strainer (Flowmi Tipstrainers, VWR) into ice-cold PBS. Cells were collected by centrifugation at 300 g for 5 minutes at 4 °C before resuspension in Red Blood Cell lysis buffer (Merck) for 5 minutes, then centrifuged at 200 g for 5 minutes at 4 °C before resuspension in PBS containing 0.04% UltraPure BSA (AM2616, ThermoFisher Scientific), and finally strained through a 40-µm cell strainer to further remove cell clumps and large fragments. Cell number and viability was measured for the biopsy using Luna Cell counter as previously published^{63 (link)}. Libraries for scRNAseq were generated using the Chromium Single Cell 5′ library and Gel Bead & Multiplex Kit from 10x Genomics. We aimed to profile 5000 cells per library if sufficient cells were retained during dissociation. All libraries were sequenced on Illumina NextSeq until sufficient saturation was reached. After quality control, raw sequencing reads were aligned to the human reference genome GRCh38 and processed to a matrix representing the UMIs per cell barcode per gene using CellRanger (10x Genomics, v3.1).