Foxp3 buffer set

Single cell suspensions of bone marrow and spleen depleted of red blood cells and cell cultures (see below) were and stained extracellularly with combinations of the following antibodies coupled to PE, PerCP, APC, and/or biotin [plus streptavidin APC (Tonbo)]. Bone marrow and IL-7 cultures: B220 (eBioscience), CD43 (BD Pharmingen), IgM (BD Pharmingen), CD25 (Tonbo), and CD93 (eBioscience). Spleen and LPS cultures: B220 (eBioscience), CD21 (BD Pharmingen), CD23 (BioLegend), CD93 (eBioscience), and CD138 (BD Pharmingen). For intracellular staining, cells were first stained extracellularly with combinations of the above antibodies, then stained intracellularly with antibodies against IRF4 (Invitrogen), IRF8 (eBioscience), IgM (BD Pharmingen), VpreB (Biolegend) or isotype control using the FoxP3 Buffer Set (eBioscience). Samples were run on a FACS Calibur (Becton Dickinson) and analyzed with FlowJo software (TreeStar).

All staining experiments were performed at 4°C for 30 minutes. Antibodies used were CD3-PerCPCy5.5, CD8-APCCy7, CD25-APC, CD134-PE, TNF-α-PECy7, CD154-APC ((Becton Dickinson (BD) Biosciences)), CD4-Alexa Fluor 700, IFN-γ-eFluor450, IL2-PerCPeFluor710, Streptavidin-Alexa Fluor 700 (eBioscience), FoxP3-Alexa Fluor 488, CD25-Brilliant Violet 421 (BioLegend), CD39-biotin, CD127-PE (Miltenyi biotec), Streptavidin-ECD, CD45RO-ECD (Beckman Coulter). LIVE/DEAD fixable aqua staining kit (Life technologies) was used to discriminate live and dead cells. For intracellular staining, FoxP3 buffer set (eBioscience) was used.

Single-cell suspensions were prepared from the thymus by manual disruption using syringe plunger. PBS57-CD1d1 tetramer was obtained from the National Institutes of Health Tetramer Core Facility. The complete list of surface antibodies used is as follows: Anti-CD5 (53-7.3), anti-CD6 (BX222), anti-Ly6c (AL-21), anti-Vβ2 (B20.6), anti-Vβ7 (TR310), anti-Vβ8.1-8.2 (MR5-2), anti-Vβ8.3 (1B3.3), anti-Egr2 (erongr2), anti-CD24 (M1/69), anti-CD69 (H1.2F3). From BD Biosciences: anti-TCRβ (H57-597), anti-CD8α (53-6.7); from BioLegend: anti-CD44 (IM7); anti-CD4 (RM4-5). Surface antibody staining was done then cells were fixed and permeabilized using the FoxP3 buffer set (eBioscience). Fixed and permeabilized cells were incubated with intracellular antibodies including anti-PLZF (Mags.21F7; eBioscience), anti-Tbet (4B10; BioLegend), and anti-Rorγt (Q31-378; BD Biosciences). Cells were analyzed on a BD LSRFortessa (BD Biosciences) and data were processed with FlowJo software (TreeStar).

Cells from thymus and cLN were surfaced stained with Fixable Viability Dye eFluor506 (eBioscience), anti-CD16/CD32 Fc block (clone 2.4G2; BD Biosciences), anti-CD3-eFluor450 (clone 17A2; eBioscience), anti-CD4-FITC (clone GK1.5; BD Biosciences or eBioscience), anti-CD25-PercPcy5.5 (clone PC61; BD Biosciences) for 20 min. Next, cells were permeabilized for 30 min, followed by 30 min of intracellular staining for anti-Foxp3-PE (clone FJK-16s; eBioscience). For the intracellular staining, the Foxp3 buffer set (eBioscience) was used and all incubation steps were done on ice.

Enriched CD4+ T cells were processed for HIV-Flow assay^{50 (link)}. Briefly, after 1 h pre-incubation with 5 μg/ml Brefeldin A (BFA) and 24 h stimulation with of 162 nM PMA and 1 µg/ml ionomycin in the presence of ARVs (200 nM lamivudine and 200 nM raltegravir), extracellular staining was performed using the following antibodies: Live/Dead Aqua Cell Stain (ThermoFisher Scientific cat.L34957), CD45RA APC-H7 (clone HI100; BD cat.560674), CCR7 BB700 (clone 3D12; BD cat.566437), PD-1 BV605 (clone EH12.2H7; Biolegend cat.329924), TIGIT eF450 (clone MBSA43; eBioscience cat.48-9500-42), HLA-DR AlexaFluor700 (clone G46-6; BD cat.560743), ICOS BV785 (clone C398.4 A; Biolegend cat.313534), α4/CD49d PE-Cy7 (clone 9F10; Biolegend cat.304313) and β1/CD29 BB515 (clone MAR4; BD cat.564565). Cells were simultaneously fixed and permeabilized with the FoxP3 Buffer Set (eBioscience), followed by intracellular staining of HIV p24 with clone 28B7 APC (MediMabs cat.MM-0289-APC) and clone KC57 PE (Beckman Coulter cat.6604667). All samples were resuspended at a final concentration of 1 × 10⁶ cells/ml in PBS and filtered prior to cell sorting.