Staining of samples for flow cytometry was conducted as described previously (
Liang
et al., 2020 ). Briefly, to distinguish live cells, samples were incubated in a fixable yellow viability dye (Invitrogen, # L34959) for 30 minutes, followed by 30 minutes incubation in a mix of directly conjugated antibodies and Fc block (see
Table 4 for antibody panel and concentrations). Following centrifugation, the staining mix was removed and remaining pellets were incubated for 5 minutes in 4% paraformaldehyde (PFA) for fixation. Once fixed, samples were centrifuged, PFA removed and pellets resuspended in 200μl of FACS buffer. Flow cytometry was conducted on a BD Fortessa at the NIHR BRC flow core facility at King’s College London, with compensation controls employed as described previously. All analysis was carried out using
FlowJo version 10.6.0 software (see
Extended Figure 1 for gating strategies employed (
Hore
et al., 2021a )).
et al., 2020
et al., 2021a