Negative magnetic bead selection kit

As previously described,11 (link) anaesthetised mice were mounted on an IPL system, and after exsanguination and thoracotomy, their pulmonary artery and left atrium cannulated. Lung perfusion with supplemented RPMI-1640 and ventilation was commenced (see online supplementary figure E1). After an initial washout period, lungs underwent either 15 min of open perfusion (compartment protocol, see online supplementary figure E2A), 2 hours of ischaemia/2 hours of reperfusion (I/R protocol, see online supplementary figure E2B) or adoptive transfer of blood monocytes prior to I/R (adoptive transfer protocol, see online supplementary figure E2C).
For monocyte depletion experiments, mice received intraperitoneal clodronate liposomes (Formumax Scientific, Palo Alto, California, USA) 24 hours prior to the I/R protocol. For adoptive transfer experiments, monocytes were isolated from blood of donor mice using a negative magnetic bead selection kit (StemCell Technologies, Grenoble, France) and infused into the IPL.

CD8⁺ T cells were isolated from naive spleens using a negative magnetic bead selection kit (Biolegend). MDSCs were isolated from the spleens of mice bearing 4T1 tumours that had either been treated with vehicle or PI-3065 using a negative magnetic bead selection kit (Stemcell). CD8⁺ T cells were labelled with Tag-It-Violet (Biolegend) prior to stimulation with plate-bound anti-CD3 10 μg/ml (Biolegend, 145–2C11), 1 μg/ml soluble anti-CD28 (Biolegend, 37.51) and 30 IU/ml IL-2. MDSCs were added to the CD8⁺ T cells at a ratio of either 1:1 (MDSCs:CD8) or 4:1. Cocultures were left for 3 days prior to flow cytometric analysis.

Human primary PBMCs were obtained via peripheral phlebotomy as described above and resting CD4⁺ T cells were isolated using a magnetic bead negative-selection kit from StemCell Technologies according to the manufacturer’s instructions. Ex vivo cell culture assays with resting CD4⁺ T cells from HIV-1-infected aviremic participants were performed as previously described (38 (link), 39 (link)). Briefly, cells were cultured at a concentration of four million cells/ml of culture medium per condition. Cells were exposed to IL-15 (100 ng/ml), followed by the addition of benzotriazine analogues (100 μM), and then cultured for 48 h. Cells then underwent centrifugation, and cell-associated RNA was extracted. RNA isolation and purification were performed using TRIzol RNA isolation according to the manufacturer’s instructions. For the detection of HIV-1 mRNA transcripts, qPCR was performed on cell-associated RNA isolated from cultured cells, as described previously (38 (link), 39 (link)).