Live dead fixable far red

A CD8⁺ T-cell line reactive to the HLA-A2-restricted peptide Influenza virus matrix protein MP_58-66 (32% peptide-specific by tetramer staining) was thawed and rested for 3 h before use. Meanwhile, islet clusters were dissociated with TrypLE, stained with 1 μM CFSE or CellTrace Violet (CTV) and incubated for 2 h with 0.1 μM Influenza MP_58-66 or peptide diluent, respectively. After washing, CFSE- and CTV-labelled SC-islets were mixed in equal numbers and cultured for 6 h in triplicate at 1 × 10⁵ cells/each per well in 96-well flat-bottom plates, alone or with increasing numbers of T cells (0.2 × 10⁵, 1 × 10⁵, 5 × 10⁵, and 10 × 10⁵), corresponding to effector-to-target (E:T) ratios of 1:5, 1:1, 5:1, and 10:1, respectively. After washing, cells were stained with Live/Dead Fixable Far Red (Thermo Fisher), antibodies against HLA-A, B, C (RRID: AB_2566151) and PD-L1 (RRID: AB_940368) and acquired on a BD LSRFortessa flow cytometer. SC-islets were analysed by FlowJo after gating on Live/Dead¯ events and separation of CFSE⁺ (peptide-pulsed) and CTV⁺ (unpulsed) populations. Percent peptide-specific lysis at different E:T ratios is expressed as the ratio of live CFSE⁺/CTV⁺ cells normalized to the same ratio in wells containing SC-islet targets alone.

Bone marrow derived neutrophils were isolated from C57/B6 mice using EasySep Mouse Neutrophil Enrichment Kit (STEMCELL, 19762) following manufacturer protocol. Metacyclic promastigotes were labelled with Carboxyfluorescein succinimidyl ester (CFSE) by incubating for 15 minutes at room temperature followed by wash. Isolated neutrophils were infected with CFSE stained metacyclic promastigotes at multiplicity of infection (MOI) of 3 metacyclic promastigotes per neutrophil. The infection was performed in the presence of tHDL and bHDL for four hours. After 4 hours, infected neutrophils were stained with Live-dead Fixable far red (ThermoFisher, L10120) and Ly6-G PE (BD Biosciences, 551461), fixed with PFA and assayed by flowcytometry.

Human PBMC were stained in PBS with LIVE/DEAD Fixable Far Red (Thermofisher Scientific) for 15 min at room temperature. Human Fc‐block (BD Biosciences) was then added at 500 ng/test for a further 15 min at room temperature. Cells were washed once and stained in PBS + 2% FBS for 30 min at room temperature with anti‐surface marker mAb as listed in Supplementary table 1 and human MR1‐5‐OP‐RU or MR1‐Ac‐6‐FP BV421 tetramer (streptavidin‐BV421 from Biolegend). Cells were then washed twice before avidin and biotin blocking (Dako). Cells were then stained for a further 30 min at room temperature with human CD1d‐PBS44 BV605 tetramer (streptavidin‐BV605 from BD Horizon). Cells were washed twice and subsequently either subjected to intracellular transcription factor staining (see below) or fixed with 2% paraformaldehyde for 10 min at room temperature. Cells were analyzed immediately by flow cytometry using an LSRFortessa (BD Biosciences).