Anti human trustain fcx

Single cell suspensions from both mice and human samples were first stained with Zombie Violet (BioLegend, Cat#423114) and then washed 2x with PBS prior to incubation with anti-mouse TruStain FcX™ (BioLegend, Cat#101319) or anti-human TruStain FcX™ (BioLegend, Cat# 422301), respectively, to block nonspecific Fc domain binding. Cells were then stained with the desired antibodies listed in Supplementary Tables 1, 2 and washed 2x with PBS. For intracellular TLR7 staining, cells were fixed and permeabilized using Cyto-Fast™ Fix/Perm Buffer Set (BioLegend, Cat#426803), followed by staining with the anti-mouse TLR7 monoclonal antibody (Supplementary Tables 1, 2). To quantitate the functional subset of FRβ expressing cells, a folate receptor-targeted fluorescent dye conjugate (folate-Cy5, synthesized in-house; or folate-fluorescein, MedChemExpress, #910661-33-5) were used. After washing, cells were resuspended in FACS buffer and examined using an Attune NxT flow cytometry prior to data analysis using Attune Cytometric Software or FlowJoucodep™ v10.

Patients’ peripheral blood mononuclear cells were stained with 5 uL of fluorochrome-conjugated anti-CD3 (HIT3a), anti-CD4 (OKT4), and anti-CD8 (SK1) for 30 min at 4 °C after blocking with anti-human TruStain FcX (BioLegend) in a fluorescent activated cell-sorting buffer (PBS containing 1% BSA and 0.1% sodium azide). Data acquisition used a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA) and analysis was carried out using FlowJo (v.10.5.3, TreeStar, Woodburn, OR, USA).

Single cell suspensions were washed once with PBS and stained with 100 μL FVD (eBioscience) diluted in PBS for 30 min at 4°C. Cells were washed with equal volume of PBS/2% FCS, followed by Fc blocking with 100 μL anti-human TruStain FcX (BioLegend) in PBS/2% FCS for 10 min at 4°C. Cells were then stained with the PerCP/Cy5.5 anti-CD45 (Biolegend) for 30 min at 4°C and were washed with equal volume of PBS/2% FCS and resuspended in 200 μL PBS/2% FCS for FACS analysis. Data were acquired using an LSR II (BD Biosciences) and analyzed using FlowJo v.10.1 software (TreeStar).