Pma ionomycin cocktail

Dimyristoylphosphoglycerol (DMPG), Dioleoylphosphatidylethanolamine (DOPE), and Dimyristoylphosphatidylcholine (DMPC) were purchased from Avanti Polar Lipid (Alabaster, USA). Cholesterol was purchased from Sigma-Aldrich (Steinheim, Germany). VEGFR-2 peptides, V1 (YMISYAGMV, purity > 99.15%), V2 (YTVILTNPI, purity > 97.30%), and V3 (IQAANVSAL, purity > 96.03%) were synthesized by China Peptides Co. (Shanghai, China). 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) was purchased from Invitrogen (Carlsbad, CA). Flow cytometry antibodies, PMA/ionomycin cocktail, and IFN-γ and IL-4 ELISA kit (ELISA MAX^™ Deluxe), were purchased from BioLegend (San Diego, CA). All of the solvents and reagents were molecular grade.

For the flow cytometric analysis experiments, 500,000 PBMCs or 250,000 monocytes were resuspended in Aim V serum-free media (Gibco) and were added to 48-well plates. The cells were then stimulated with exosomes from healthy donors and sarcoidosis patients isolated from 1, 5 and 15 ml of BALF. As reported in Fig. 1C, these BALF volumes typically correspond to 7.3; 36.5; and 146 × 10⁶ vesicles/well for patients. Negative control was PBS added in equal volume as the PBS-suspended vesicles, positive controls were PMA/Ionomycin cocktail used at 1× dilution (Biolegend), Concanavalin A or 100 ng/ml LPS. To block cytokine release for the intracellular stainings, Monensin (Biolegend) used at 1× dilution according to manufacturer’s protocol was added after 2 h of stimulation. After 6 h of stimulation, plates were spun down at 350 × g for 10 min and cells were resuspended in ice cold PBS + 0.5 mM EDTA and incubated on ice for 5 min to detach cells, before transferring them to 96 well plates for analysis.

Thawed PBMCs were rested in R10 overnight at 37°C in 5% CO₂. Cells were stimulated with Gag, Env, and Nef pools of overlapping HIV-1 clade C consensus peptides (NIH AIDS Reagent Program) at a final concentration of 2 μg/ml for each peptide in the presence of anti-CD28/anti-CD49d at 1 μg/ml (BD) and anti-CD107a (PE-Cy7, H4A3; Biolegend). PMA/Ionomycin cocktail (Biolegend) and 0.2% DMSO were used as a positive and a negative control, respectively. Cells were incubated for 6 h and Brefeldin A at 5 μg/ml (Biolegend) and Monensin at 1 μg/ml (Biolegend) were added after 1 h of stimulation outset. Stimulated cells were washed and stained with Live/Dead near-IR stain (Invitrogen) for 30 min at room temperature. Then, cells were washed again and incubated for another 30 min at 4°C with antibodies against CD3, CD4, and CD8 as above. Cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD) for 45 min at 4°C and stained with antibodies against IFN-γ (PE-Dazzle594, B27; Biolegend), IL-2 (BV510, MQ1–17H12; Biolegend), TNF-α (AlexaFluor700, MAb11; Biolegend), MIP-1β (PE, D21–1351; BD, San Diego, California, USA), Perforin (FITC, B-D48; Biolegend), and Granzyme B (APC, GB11; Invitrogen) for 30 min at 4°C. Cells were washed and acquired on a BD LSR II.