Anti cd45 af700

For FCGRs detection, primary B cells, monocytes, and macrophages were incubated with fluorochrome-labeled antibodies specific for humans before fixation: FITC mouse anti-human CD32 (552883; BD Pharmingen, San Diego, CA, USA), APC mouse anti-human CD64 (561189; BD Pharmingen, San Diego, CA, USA ), CD16 Rabbit PAb (16559-1-AP; Proteintech,--). FITC-anti-Rabbit IgG (H+L) (SA00003-2; Proteintech, Chicago, IL, USA) was used as the secondary antibody for CD16.
For SARS-CoV-2 infected primary immune cells, surface staining was conducted before fixation with AF700-anti-CD45 (368514; BioLegend, San Diege, CA, USA), BV650-anti-CD11b (101239; BioLegend, San Diege, CA, USA), PE-anti-CD68 (333808; Biolegend, San Diege, CA, USA), and Percp Cy5.5-anti-CD14 (367110; Biolegend, San Diege, CA, USA). Antibody stained cells were fixed overnight with 4% PFA at 4 °C and taken out of the BSL3 lab for downstream analysis. Cells were stained further with in house made SARS-CoV NP pAb (1:500) at 4 °C for 30 min after permeabilization. Then, cells were stained with FITC-anti-Rabbit IgG (H + L) (SA00003-2; Proteintech, Chicago, IL, USA) at room temperature for 30 min.

1–2 × 10⁶ cells were placed in 200 uL of PBS1X with 2% FBS on ice and antibodies were added, mixed, and incubated for 30 min. Then, cells were washed with PBS1X and centrifuged at 800×g for 5 min. Finally, samples were acquired in a CytoFLEX LX cytometer (Beckman Coulter). Analysis was performed using CytExpert software. The following anti human antibodies were used: AF488 anti-GRP78 (Clone: C38), AF700 anti-CD45 (Clone: HI30), APC anti-CD34 (Clone: 561), PE/Cy7 anti-CD38 (Clone: HB-7), PerCP/Cy5.5 anti-CD19 (Clone: HIN19), PE/Dazzle anti-CD10 (Clone: HI10a), Brilliant Violet 421 anti-CD184 (clone: 12G5) and the Zombie Aqua™ Fixable Viability Kit was used according to the manufacture (Biolegend).

Lung MAITs were expanded ex vivo using a previously published method for the expansion of human MAIT cells (Liu et al., 2020 (link)). Briefly, lung homogenates from healthy WT mice were cultured for 14 days in the presence of mouse recombinant IL-2 and sulfate latex beads of 5-OP-RU/MR1 artificial antigen-presenting cells. To isolate monocytes, lung cells from healthy WT mice were surface stained with anti-CD45 AF700 (BioLegend), anti-CD11b-FITC (BioLegend), anti-Siglec-F- BV711 (BD), and anti- Ly6C (PE dazzle 594) for 30 min at 4°C. We obtained CD45⁺, Siglec-F^-, Ly6C⁺, CD11b⁺ monocytes by flow sorting on a FACS Aria II (BD, Franklin Lakes, NJ). We incubated 20,000 MAITs with 10,000 monocytes for 18 hr, in a total volume of 200 µL in 96-well U-bottom plates. MAIT-monocyte co-culture was stimulated with 200 nM 5-A-RU and 50 µM MeG (Li et al., 2018 (link)) in RPMI 1640 with 10% FBS with 1% penicillin/streptomycin and 1% HEPES. MAIT cells that were stimulated/unstimulated and monocytes that were stimulated/unstimulated were kept as controls. After 18 hr, the cell supernatant was collected and stored at −20°C. We measured IFN-γ and GM-CSF levels in supernatants using ELISA as per manufacturer’s instruction in BioLegend ELISA MAX standard sets for mouse IFN-γ and mouse GM-CSF.