The following antibodies were used for surface staining at 4°C: anti-CD4 (Biolegend, 1:200, RM4-5)12 (link), anti-ICOS (Biolegend,1:200, 15F9)12 (link), anti-CD19 (Biolegend,1:200, 6D5)12 (link), anti-CXCR5 biotin (BD Biosciences,1:100, 2G8)12 (link), GL7 (BD Biosciences,1:200, GL-7)12 (link), CD69 (Biolegend,1:200, H1.2F3)12 (link), and anti-IA (Biolegend,1:200, M5/114.15.2)12 (link). For further CXCR5 detection, streptavidin-BV421 (Biolegend, 1:400, 405225) was used at 4°C. For intracellular staining, samples were fixed with the Foxp3 Fix/Perm buffer set according to the manufacturer’s instructions (eBioscience). Samples were then intracellularly stained with anti-IgG1 (BD Biosciences, 1:200, A85-1)12 (link), anti-FoxP3 (eBiosciences, 1:200, FJK-16S)12 (link), anti-Ki67 (BD Biosciences, 1:100, B56)12 (link), anti-Glut1 (Abcam, 1:200, EPR3915)31 (link), anti-Shmt2 (Abcam, 1:200, ab64417,)32 (link) or anti-Shmt1 (Novus Biologicals, 1:200, NBP2-32173,)32 (link) at 4°C. In some cases, a donkey anti-rabbit BV421 secondary was used (Biolegend, 1:400, 406410)
Protocol detail
Multiparameter Immune Cell Phenotyping
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anti-CD4
anti-ICOS
anti-CD19
anti-CXCR5 biotin
anti-IA
streptavidin-BV421
Foxp3 Fix/Perm buffer
anti-IgG1
anti-FoxP3
anti-Ki67
anti-Glut1
anti-Shmt2
anti-Shmt1
Antibodies
Biologicals
Biotin
Buffer
Cxcr5
Donkey
Glut1
Icos
Igg1
Intracellular
Novus
Perm
Rabbit
Streptavidin
Corresponding Organization : Harvard University
Other organizations : Dana-Farber Cancer Institute, Broad Institute, Center for Systems Biology
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Variable analysis
independent variables
- Anti-CD4 (Biolegend, 1:200, RM4-5)
- Anti-ICOS (Biolegend,1:200, 15F9)
- Anti-CD19 (Biolegend,1:200, 6D5)
- Anti-CXCR5 biotin (BD Biosciences,1:100, 2G8)
- GL7 (BD Biosciences,1:200, GL-7)
- CD69 (Biolegend,1:200, H1.2F3)
- Anti-IA (Biolegend,1:200, M5/114.15.2)
- Streptavidin-BV421 (Biolegend, 1:400, 405225)
- Anti-IgG1 (BD Biosciences, 1:200, A85-1)
- Anti-FoxP3 (eBiosciences, 1:200, FJK-16S)
- Anti-Ki67 (BD Biosciences, 1:100, B56)
- Anti-Glut1 (Abcam, 1:200, EPR3915)
- Anti-Shmt2 (Abcam, 1:200, ab64417)
- Anti-Shmt1 (Novus Biologicals, 1:200, NBP2-32173)
- Donkey anti-rabbit BV421 secondary (Biolegend, 1:400, 406410)
- Not explicitly mentioned
- Surface staining at 4°C
- Intracellular staining after fixation with Foxp3 Fix/Perm buffer
- Not explicitly mentioned
- Not explicitly mentioned
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