Il 10 apc cy7

Draining s.c. LNs (axillary and popliteal) and the spleen were isolated from mice. Spleens were first mashed into a single cell suspension with plain DMEM media (Gibco 11966025), filtered through 70 μM cell strainers, and lysed with ACK lysis buffer (Gibco A1049201). LNs were digested with 1 mg/mL Ca²⁺ supplemented Collagenase D (Roche 11088866001) for 45 min at 37°C and gently mashed into a single cell suspension, also with DMEM and through 70 μM cell strainers. The cells were resuspended in IMDM media (Gibco 12440053), supplemented with 10% FBS and 1% Penicillin-Streptomycin (Gibco 15140122), and counted using a LUNA automated fluorescent cell counter (Logos biosystems). Cells were seeded at a count of 1x10⁶ - 3x10⁶ per well in 96-well round-bottom plates for subsequent antibody staining for flow cytometry. Antibodies against the following markers were used: CD3 – BUV395 (BD Biosciences 563565), CD8-BUV737 (BD Biosciences 612759), CD4 – BUV496 (BD Biosciences 612952), Foxp3 – FITC (BD Biosciences 560403), CD25 – BV605 (BioLegend 120235), ST2 – BV421 (BD Biosciences 566309), Lag3 – PerCP-Cy5.5 (BD Biosciences 564673), CTLA4 – PE-Cy7 (eBioScience 17-1522-82), IFNγ – APC (BioLegend 505810), TNFα – BV605 (BioLegend 506329), IL-2 – FITC (BioLegend 503806), IL-10 – APC-Cy7 (BioLegend 505036), PD-1 – BV711 (BioLegend 135231), Tim3 – PE (BD Biosciences 566346).

Single‐cell suspensions were stained with a mixture of 5 to 6 fluorophores including and 7‐aminoactinomycin D (7‐AAD) for viability and acquired on the BD Verse flow cytometer with data analyzed using FlowJo software (BD Biosciences). Human B‐cell subsets were identified using the following antibodies: CD19‐APC, CD27‐BV510, CD38‐APC‐Cy7, CD24‐BV421, and IL‐10‐PE‐Cy7 (all from BioLegend). Mouse B‐cell subsets were identified using the following antibodies: B220‐PE, CD23‐APC, CD21‐PE‐Cy7, IgM‐BV421, CD138‐APC, CD86‐PE‐Cy7, CD69‐APC‐Cy7, and IL‐10‐APC‐Cy7 (all from BioLegend). Intracellular IL‐10 expression was detected as previously reported.³³ Immunophenotype of human and murine B‐cell subsets analyzed are listed in Supplementary Table 1.