Brilliant violet 421 cd25

CBMCs and maternal PBMCs were thawed, aliquoted at 1 × 10⁶ cells, cells were washed, stained, fixed, and permeabilized, as per the manufacturer’s instructions (FoxP3 Fix/Perm kit; eBiosciences) using standard protocols using the following antibodies: APC/Cy7 CD3 (clone OKT3), PerCP CD4 (clone RPA-T4), Brilliant Violet 421 CD25 (clone BC96), Brilliant Violet 650 CD127 (clone A019D5), Brilliant Violet 605 CD45RO (clone UCHL1), FITC CCR7 (clone G043H7), Brilliant Violet 510 CD8 (clone SK1), Brilliant Violet 510 CD14 (clone M5E2), Brilliant Violet 510 CD19 (clone HIB19, BioLegend), FITC Ki67 (clone Ki67, BD Pharmingen), PE FoxP3 (clone PCH101, eBioscience), and LIVE/DEAD aqua amine (Invitrogen). Flow cytometry data were collected on an LSR II four-laser flow cytometer (BD) with FACSDiva software. Color compensation was performed using compensation beads. Fluorescence-minus-one samples were used to define negative and positive populations. Cellular profiles were gated on live, single-cell, dump negative (CD14, CD19, CD8), CD3 + lymphocytes.

Alexa Fluor 488 NK1.1 (PK136), Biotin-CD4 (GK1.5), Alexa Fluor 488-CD4 (GK1.5), APC-eFluor 780-CD4 (GK1.5), FITC-CD3ε (17A2), Alexa Fluor 647-CD3ε (17A2), Alexa Fluor 488-CD206, Brilliant Violet 421-CD3ε (17A2), FITC-F4/80, Alexa Fluor 488-CD3ε (145-2C11), Alexa Fluor 647 CD49b (DX5), Pacific Blue-CD49b (DX5), PE-NK1.1 (PK136), Brilliant Violet 421 NK1.1 (PK136), PE/Cy5-CD4 (GK1.5), APC-TCRγδ (GL3), Biotin-IFN-γRα (2E2), Brilliant Violet 421-CD25 (PC61), PE-CD25 (PC61), Biotin-CD122 (5H4), PE/Cy7-IFN-γ (XMG1.2), PE-GM-CSF (MP1-22E9), Pacific Blue-TNF-α (MP6-XT22), PercCP/Cy5.5-CD69, Biotin-BrdU (Bu20a), FITC-Ki67 (16A8), Alexa Fluor 647-streptavidin, APC-Cy7-Streptavidin, PE-Cy7-Streptavidin, Brilliant violet 421-CD62L (MEL-14) and rabbit polyclonal anti-Asialo-GM1 (aGM) antibodies were purchased from Biolegend (San Diego, CA). APC-RORγt (AFKJS-9), APC-T-bet (4B10) and PE/Cy7-T-bet (4B10) were procured from eBioscience (San Diego, CA). Anti-NK1.1 (PK136), anti-F4/80 (Cl:A3–1), anti-Gr1 (RB6-8C5) and isotype control antibodies for in vivo use were procured from BioXcell (West Lebanon, NH). Alexa Fluor 647-labeled CD1d tetramer (mCD1d, PBS-57) and unloaded control tetramer were received from NIH Tetramer Core Facility (Atlanta, GA). 5-Bromo-2′-deoxyuridine (BrdU) was purchased from Sigma-Aldrich.

Single cell suspensions of liver, spleen, bone marrow and peripheral blood samples were prepared from wild type (WT) and Sptbn1^+/- mice. Mice were euthanatized with CO₂ and their liver, spleen, bone marrow, and peripheral blood were collected. Livers and spleens were minced with scissors and a razor knife and then filtered with 40 μm nylon mesh strainer (BD Biosciences, San Jose, CA). All single suspended cells were depleted of red blood cells using 1×RBC lysis buffer for 5 minutes on ice. Single cells were suspended in Dulbecco's PBS (D-PBS) (Mediatech, Inc.) and stained with fluorescence-conjugated antibodies: PE-Cy7-CD45, APC-CD4, APC-F4/80, Alexa Fluor® 488-Ly-6G/Ly-6C (Gr-1), Brilliant Violet 421-CD11b, Brilliant Violet 421-CD25, and Alexa Fluor® 488-FOXP3 (Biolegend). Subsequent to being washed, cells were analyzed using a BD LSRII flow cytometer (BD Biosciences, San Jose, CA). Profiles of lymphoid and myeloid cells in CD45-gated leukocytes were assessed using FlowJo7 software (FlowJo, Williamson Way, Ashland, OR).