Flow Cytometry Using the 7-AAD Cell Viability Staining

Lymphocyte viability (Saade et al., 2012) was assessed with the use of flow cytometry using the fluorescent DNA intercalator 7-AAD (7-aminoactinomycin). 7-AAD is a fluorescent derivative of actinomycin D that selectively binds to GC regions of the DN, exposed during cell death; and is frequently used to stain and exclude dead cells in flow cytometry (Zembruski et al., 2012).

Cryopreserved PBMCs were thawed and rested in R10 media at 37°C for 3 h. Cells (2−3 x 10⁵) were incubated for 18 h with antimicrobials tested. After incubation, cells were stained with CD3 Alexa Fluor 700 and CD4 PerCp Cy5.5 (BD Pharmingen) and CD8-APC AF750 (Invitrogen) for 30 min at 4°C, then washed with FACS buffer (PBS containing 1% bovine serum albumin and 0.05% sodium azide). After washing, PBMCs were stained with 7-AAD (BD Pharmingen) for 15 min. Cell events were acquired on a LSRII flow cytometer and FlowJo software was used to analyse FCS files. The gating strategy is detailed in Supplementary Figure S3.

This study was approved by both the Austin Health ethics committee and the Human Research Ethics committee of the University of Cape Town. The investigators obtained written informed consent from the participants.

We sought to determine the maximal antimicrobial concentrations for cell viability using the cytotoxicity based LDH assay and the 7-AAD cell viability staining for commonly implicated antimicrobials in severe T-cell mediated hypersensitivity—ceftriaxone, flucloxacillin, piperacillin-tazobactam, isoniazid, and rifampicin.