Foxp3 transcription factor kit

Flow cytometry was used to assess the following major cell lineages: CD8⁺ T cells, conventional CD4⁺ T cells, regulatory T cells, B cells, myeloid cells (monocytes, dendritic cells and plasmacytoid dendritic cells), natural killer cells and granulocytes. Staining was performed as per preparation for cell sorting described above, with the addition of fixation and permeabilization for intracellular staining with the Foxp3/Transcription Factor kit (eBioscience). The antibodies and viability dyes used are described in the Key Resources Table.

We thawed PBMCs from cryovials and washed them as described above, and we seeded 2.5 × 10⁶ cells/ml in RPMI 1640 with 10% FBS or 2% Phx. We stimulated PBMCs with 10 multiplicities of infection (MOIs) of strain 1100–2 fixed E. coli for 20 h and blocked extracellular transport with brefeldin A for a total of 4 h. We performed extracellular staining with fixable viability dye eFluor 780 (eBioscience), anti-CD3-BUV395 (BD Biosciences), anti-CD8-BV605 (BioLegend), anti-CD4-BV510 (BioLegend), anti-Vα7.2-PE-Cy7 (BioLegend), anti-LAG-3-BV786 (BioLegend), anti-CD25-BV650 (BioLegend), anti-PD-1-PerCPCy-5.5 (BioLegend), anti-CD161-PE/Dazzle-594 (BioLegend), anti-CD69-PE-Cy5 (Invitrogen), anti-CD161-allophycocyanin (BioLegend), and anti-human PE-MR1–5-OP-RU tetramer (NIH Tetramer Core Facility). The PBMCs were fixed and permeabilized using an Foxp3/transcription factor kit (eBioscience) and stained intracellularly with anti–granzyme B Alexa Fluor 700 (BioLegend), anti-TNF-α-eFluor-450 (Invitrogen), anti-IFN-α-FITC (BioLegend), and anti-T-bet-BV711 (BioLegend). We collected data using a five-laser LSRFortessa flow cytometer (BD Biosciences), and the flow data were analyzed using FlowJo software version 10 (BD Biosciences).

We cultured 2,000–5,000 MAIT cells from MAIT cells expanded in RPMI 1640 with 10% FBS or 2% Phx with 10,000–25,000 THP-1 cells to serve as APCs in RPMI with 10% FBS without cytokines overnight. We then stimulated the coculture of MAIT cells and THP-1 with 10 MOIs of strain 1100–2 fixed E. coli for 20 h and blocked extracellular transport with brefeldin A for a total of 4 h. For extracellular staining, we used Zombie ultraviolet fixable viability dye (BioLegend), anti-CD3-BUV395 (BD Biosciences), anti-CD8-BV605 (BioLegend), anti-CD4-BUV496 (BD Biosciences), anti-Vα7.2-PE-Cy7 (BioLegend), anti-LAG-3-BV786 (BioLegend), anti-CD69-BUV563 (BD Biosciences), anti-CD161-PE-Dazzle-594 (BioLegend), anti-TIM-3-BV421 (BioLegend), anti-CD25-BV650 (BD Biosciences), and anti-human PE-MR1–5-OP-RU tetramer (NIH Tetramer Core Facility). The cells were fixed and permeabilized using Foxp3/transcription factor kit (eBioscience) and stained intracellularly with anti-granzyme-B Alexa Fluor 700 (BioLegend), anti-TNF-α eFluor 450 (Invitrogen), and anti-IFN-γ-FITC (BioLegend). Sample data were acquired with a five-laser Cytek Aurora flow cytometer (Cytek) and analyzed using FlowJo software version 10 (BD Biosciences).

Cells were analyzed for cell-surface markers using fluorophore-conjugated antibodies (BioLegend, eBioscience). Cell surface staining was performed in 1X PBS and intracellular staining was performed using the eBioscience Foxp3/Transcription Factor kit. Flow cytometry was performed using the Attune NxT and data were analyzed with FlowJo software (BD). Cell surface and intracellular staining was performed using the following fluorophore-conjugated antibodies: CD45.1 (A20), CD45.2 (104), NK1.1 (PK136), CD49b (DX5), TCRβ (H57–597), CD3 (17A2), CD200r1 (OX-110), CD25 (PC61), IL-18r (P3TUNYA), IFN-γ (XMG1.2), I-A/I-E (M5/114.15.2), CD19 (6D5), CD11c (N418), CD11b (M1/70), XCR1 (Zet), Ly6G (1A8), CD88 (20/70), F4/80 (BM8), Tim-4 (RMT4–54), Ly6C (HK1.4), CX3CR1 (SA011F11), and CD9 (MZ3).

Single cell suspensions were prepared from spleens and stained with fluorochrome-conjugated antibodies. For flow cytometry of the adoptive transfer and influenza experiments, Live/Dead Zombie Aqua, anti-CD45.1-AF700 (A20), anti-CD45.2-BV421 (104), anti-CD19-BV785 (6D5), and anti-CD23 biotin (B3B4), and anti-CD11c (N418) were from Biolegend. Anti-CD43-PE (S7) was from BD Biosciences. Cells were analyzed on an LSRII, and data analyzed using FlowJo software (Tree Star). Intracellular stains for T-bet were performed with anti-T-bet-APC (4B10) from Biolegend and the Foxp3 transcription factor kit (eBioscience) according to manufacturer's instructions. For FACS sorting to isolate subsets, anti-CD43-APC (S7) was from BD Biosciences. Anti-CD23-PE Cy7 (B3B4), anti-CD21/CD35-eFluor 450 (4E3), anti-CD45R-FITC (B220, RA3-6B2), and anti-CD93 (AA4.1)-APC were from eBioscience. Stained splenocytes were analyzed with a BD FACSCanto II, or sorted using a BD FACSAria III, BD FACSAria Fusion, iCyt Reflection (Sony Biotechnology), or Beckman Coulter MoFlo. Follicular (FO) B cells were isolated as CD93 (AA4.1)^- CD43^- B220⁺ CD21/35⁺ CD23⁺. Marginal zone (MZ) B cells were isolated as CD93 (AA4.1)^- CD43^- B220⁺ CD21/35⁺ CD23^Lo. ABCs were isolated as CD93 (AA4.1)^- CD43^- B220⁺ CD21/35^- CD23^-. Analyses were done using FlowJo software.