Live dead fixable yellow viability dye

1 × 10⁶ PBMCs/well were plated in either duplicate (Myeloid Cell panel) or triplicate (Memory T Cell panel and Regulatory and γδ T Cell panel) in 96-well round-bottom plates. Cells were either overnight stimulated with ASFV-G at MOI 0.5 or incubated with media (RPMI supplemented with 2% FBS, 1× antibiotic/antimycotic, 1× non-essential amino acids (NEAA) and 1× L-glutamine) at 37 °C 5% CO₂. The following morning, staining procedures began immediately for the Myeloid Cell panel. For the two T Cell panels, brefeldin A (1:1000) (BD, Franklin Lakes, NJ, USA) was added to the unstimulated and virus-stimulated replicates, while Leukocyte Activation Cocktail with GolgiPlug (1:333) (Southern Biotech, Birmingham, AL, USA) was added as positive control replicate and incubated for another 4 h at 37 °C 5% CO₂. After being stained with LIVE/DEAD Fixable yellow viability dye (Invitrogen, Waltham, MA, USA), cells were staining with either the two T cell panels, memory T cell panel (Supplementary Table S1) and regulatory and γδ T Cell panel (Supplementary Table S2), following intracellular cytokine staining protocol or with the myeloid cell panel (Supplementary Table S3) following extracellular flow cytometry staining protocol. All plates were run on an Agilent NovoCyte 3000 with NovoSampler Pro System (violet, blue and red lasers) and data was analyzed in NovoExpress Software version 1.5.0.

Infection studies were performed using the T cell-based reporter cell line JLTRG-R5 (obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Jurkat LTR-GFP CCR5 + cells (JLTRG-R5) (Cat #11586), from Dr. Olaf Kutsch)^{46 (link),47 (link)}. Prior to infection, virus concentrations were normalized using the Lentivirus Rapid Quantitation Kit (Cell Biolabs). 1 × 10⁵ cells per well were plated into a 96 well flat-bottom plate and infected by incubating with virus for 48 h in the presence or absence of 2.5 μM saquinavir. 48 h post-infection, cells were stained with a live/dead fixable yellow viability dye (Invitrogen) and fixed in 1% PFA. Cells were analyzed for EGFP expression and viability using an LSRII flow cytometer (Becton Dickinson).