Polychromatic flow cytometry and cell sorting were performed on stained mononuclear cells as previously described15 (link). Antibodies against the following antigens were used for staining at predetermined concentrations: CD4 (clone OKT4), CD8 (SK1), IFNγ (4S.B3), IL-17 (eBio64DEC17), IL-21 (3A3-N2), IL-22 (IL22JOP), TNFα (MAb11), and PD-1 (J105) from eBioscience; CCR5 (3A9), CD3 (SP34-2), CD8 (SK1), CD16 (HI149), CD20 (2H7), CD23 (M-L233), CD45 (D058-1283), CD123 (7G3), and HLA-DR (L243) from BD; c-Kit (104D2), CD1a (HI149), CD11c (3.9), CD14 (M5E2), CD34 (561), CD95 (DX2), and IL-2 (MQ1-17H12) from Biolegend; CD127 (eBioRDR5) and CD218a (H44) from Thermo; NKp44 (2.9) from Miltenyi; and CD28 (CD28.2) from Beckman Coulter. CD4+ and CD8+ TM were defined as CD95+ singlet, clean, live, CD3+ lymphocytes. NKp44+ ILC3s were defined as CD45+ singlet, clean, live, lineage-negative (CD1a, CD3, CD8, CD11c, CD14, CD16, CD20, CD23, CD34, CD123), NKp44+CD127+CD218+ lymphocytes. Positive/negative gating based on clearly grouped populations, historical-determined expression, and the use of internal controls (Supplementary Fig. 8 ). A threshold of 100 collected events in the parent population was utilized for all subset expression analysis.
Protocol detail
Polychromatic Cytometry and Cell Sorting
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TNFα (MAb11),
PD-1 (J105)
IL-2 (MQ1-17H12)
CD127 (eBioRDR5) and CD218a (H44)
NKp44 (2.9)
CD28 (CD28.2)
Antibodies
Antigens
C kit
Ccr5
Cd123
Cd1a
Cells
Clone
Flow cytometry
Hla dr
Ifnγ
Il 17
Il 21
Il 22
Lymphocytes
Nkp44
Parent
Tnfα
Corresponding Organization :
Other organizations : National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick National Laboratory for Cancer Research
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Variable analysis
independent variables
- Antibodies against the following antigens: CD4, CD8, IFNγ, IL-17, IL-21, IL-22, TNFα, PD-1, CCR5, CD3, CD8, CD16, CD20, CD23, CD45, CD123, HLA-DR, c-Kit, CD1a, CD11c, CD14, CD34, CD95, IL-2, CD127, CD218a, NKp44, CD28
- Expression of the above-mentioned antigens on mononuclear cells
- Predetermined concentrations of the antibodies used for staining
- Gating strategy based on clearly grouped populations, historical-determined expression, and the use of internal controls
- Threshold of 100 collected events in the parent population for all subset expression analysis
- Internal controls used for positive/negative gating
- Internal controls used for positive/negative gating
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