Ghost dyetm red 710

PBMCs were cultured in 150 μl X-VIVO15 medium supplemented with 5% human AB serum (Sigma) for 20 h at 37°C in the presence of SARS-CoV-2 or CMV peptide pools (0.6 nmol/ml) and CD40 blocking antibody (Miltenyi Biotech) (0.5 ug/ml) in 96-well U-bottom plates at 2 × 10⁶ PBMCs per well. Stimulation with DW at an equal volume was performed as a negative control. After the stimulation, the cells were stained with Ghost Dye^TM Red 710 (TONBO) to discriminate viable from non-viable cells. The cells were then washed and stained with fluorochrome-conjugated surface antibodies at pre-titrated concentrations in the presence of FcR blocking (Miltenyi Biotech) for 20 min at 4°C. Antibodies used in the AIM assay are listed in Supplementary Table 1. Stained cells were fixed and permeabilized for 20 min at room temperature using the eBioscience Foxp3/Transcription Factor Staining Buffer Set. The cells were then stained with anti-IRF4 antibody (1:500 dilution) for 30 min at room temperature. After the final wash, the cells were resuspended in 200 μl PBS with 2% FBS (FACS buffer) for flow cytometry analysis. Supernatants were harvested at 20 h post-stimulation for the multiplex detection of cytokines.

After mice were anesthetized, blood and femur were collected and placed in PBS on ice. Single-cell suspensions of bone marrow were generated by mechanical disruption through a 40-μm nylon mesh (BD Bioscience). Colonic LP cells were isolated as described. Residual red blood cells were lysed using ACK lysing buffer (Invitrogen). The cells were blocked using anti-CD16/32 antibody (2.4G2 clone; Bio X Cell) for 30 min. Then, cells were stained for 30 min with antibodies for specified markers. Nonviable cells were identified using viability dye efluor 780 (eBioscience) or Ghost Dye^TM Red 710 (TONBO biosciences). Samples were run using a flow cytometer (FACSCanto; BD Biosciences, LSR-II; BD Biosciences) and analyzed using FlowJo software (Tree Star).

At each WIHS visit, PBMCs were isolated from venous blood by standardized methods, frozen, and stored in liquid nitrogen in a specimen repository. Cryopreserved PBMCs stored in liquid nitrogen were warmed in 37°C water bath, then removed and immediately diluted with 1 mL of warm cRPMI and transferred and diluted again in an additional 8mL of warm cRPMI. Samples were then washed with PBS and stained with viability reagent (Ghost Dye^TM Red 710, Tonbo Bioscience) according to manufacturer’s recommendation. Samples were stained with antibody cocktail containing CD14 (M5E2, Biolegend), CD16 (3G8, Biolegend), and dump channel: CD3 (OKT3, Tonbo Bioscience), CD19 (HIB19, Tonbo Bioscience), CD56 (5.1H11, Biolegend), CD66b (G10F5, Biolegend), gated based on living cells and excluding dump channel-positive cells. Intermediate monocytes were defined as CD14⁺CD16⁺ and non-classical monocytes were defined as CD14^dimCD16⁺. Monocytes were sorted directly into TRIzol® LS Reagent (Life Technologies) and frozen at -80°C. FCS files were exported from FACS Diva (BD Bioscience) and processed using FlowJo v10.2 (FlowJo, LLC). The quantification of intermediate and non-classical monocytes are estimated from the FACS data from all PBMC samples.