Multiparametric Flow Cytometry of MAIT Cells

Cell surface and intracellular staining was performed as previously described (15 (link), 47 (link)). Antibodies used: anti-CD3 Bv650 (OKT3, Biolegend), anti-CD3 FITC (SK7, BD Biosciences), anti-Vα7.2 PE (3C10, Biolegend), anti-CD161 Pe-Cy5 (DX12, BD Biosciences), anti-CD4 Bv711 (OKT4, Biolegend), anti-CD8 Bv570 (RPA-T8, Biolegend), anti-CD69 BUV737 (FN50, BD Biosciences), anti-CD107a BUV395 (H4A3, BD Biosciences), anti-GzB FITC (GB11, Biolegend), anti-IFNγ APC (25723.11, BD Biosciences), anti-TNF PE-Cy7 (Mab11, BD Biosciences), anti-IL17A Bv421 (BL168, Biolegend), anti-RORγt APC (R&D system), anti-RORγt Bv650 (Q21–559, BD Biosciences), anti-PLZF PECF594 (R17–809, BD Biosciences), anti-T-bet Bv711 or Bv605 (4B10, Biolegend), anti-MR1 (26.5, Biolegend), anti-HLA-DR (L243, Biolegend), anti-HLA-A2 (BB7.2, Biorad), LIVE/DEAD Fixable Aqua and Near-IR dye (Invitrogen). Flow cytometry data was acquired on BD LSRFortessa or BD Symphony A5 instruments (both BD Biosciences) and analyzed using FlowJo software v. 10.5.3 (TreeStar). Analyses of MAIT cell polyfunctionality were performed using the SPICE software v. 6.0 (48 (link)).

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Boulouis C., Gorin J.B., Dias J., Bergman P., Leeansyah E, & Sandberg J.K. (2020). Opsonization-Enhanced Antigen Presentation by MR1 Activates Rapid Polyfunctional MAIT Cell Responses Acting as an Effector Arm of Humoral Antibacterial Immunity. The Journal of Immunology Author Choice, 205(1), 67-77.

Publication 2020

anti-CD3 FITC (SK7, anti-CD4 Bv711 (OKT4, anti-CD107a BUV395 (H4A3, anti-IFNγ APC anti-IL17A Bv421 anti-RORγt APC anti-RORγt Bv650 anti-PLZF PECF594 anti-MR1 (26.5, anti-HLA-DR BD LSRFortessa BD Symphony A5

Anti cd3 Anti d Anti t Antibodies Cd161 Cell Fitc Flow cytometry Hla a2 Hla dr Ifnγ Il17a Intracellular Mait cell Rorγt Spice

Corresponding Organization :

Other organizations : Karolinska Institutet, Karolinska University Hospital, National University of Singapore

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Variable analysis

independent variables

Cell surface and intracellular staining

dependent variables

Expression of CD3, Vα7.2, CD161, CD4, CD8, CD69, CD107a, GzB, IFNγ, TNF, IL17A, RORγt, PLZF, T-bet, MR1, HLA-DR, HLA-A2

control variables

Cell surface and intracellular staining protocol previously described (15, 47)

positive controls

None specified

negative controls

None specified

Annotations

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