Multi-Parametric FACS Analysis of Tissue-Resident T Cells

Fluorescently labeled antibodies to cell surface antigens were diluted in PBS containing 1% FCS, 0.05% sodium azide, and 0.5 μg/mL CD16/CD32 (Tonbo; 2.4G2). Cells were incubated for 30 minutes at 4°C, washed, and resuspended in FACS buffer. The following antibodies were used for multi-parameter FACS analysis: CD3 (Tonbo; 17A2), CD44 (Tonbo; IM7), GITR (eBioscience; DTA-1); CD4 (RM4-5, RM4-4), CD8 (53-6.7), CD69 (H1.2F3), B220 (RA3-682), CD103 (2E7), Nrp-1(3E12), ICOS (C398.4A), and CTLA-4 (UC10-4B9) were all purchased from BioLegend, unless specified otherwise. To remove innate cells in our analysis of tissue-resident CD4⁺ T cells and tetramer analysis, antibodies directed against CD11c (N418), CD11b (M1/70), IA/IE (M5.114.15.2), HLA-DP (purified from hybridoma, B7.21), and Ly6G (1A8) were purchased from eBioscience. For HLA-DP2-CCL4/Be tetramer staining, lung cells were stained as previously described (13 (link), 17 (link)). Intracellular staining for FoxP3 was performed according to the manufacturer’s protocol (Thermo Fisher Scientific). Data were obtained using a BD FACS Canto and Celesta Flow Cytometers and analyzed using FlowJo software (v9.9.6; Treestar, Inc.).

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Atif S.M., Mack D.G., Martin A.K, & Fontenot A.P. (2022). Protective role of tissue-resident Tregs in a murine model of beryllium-induced disease. JCI Insight, 7(16), e156098.

Publication 2022

CD16/CD32 CTLA-4 (UC10-4B9) Ly6G (1A8) BD FACS Canto

Antibodies Buffer Ccl4 Cd103 Cd11b Cd4 t cells Cd44 Cell surface antigens Cells Ctla 4 Gitr Hla dp Hla dp2 Hybridoma Icos Intracellular Lung Sodium azide Tetramer Tissue

Corresponding Organization :

Other organizations : University of Colorado Anschutz Medical Campus

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Variable analysis

independent variables

Fluorescently labeled antibodies to cell surface antigens

dependent variables

Expression of cell surface antigens CD3, CD44, GITR, CD4, CD8, CD69, B220, CD103, Nrp-1, ICOS, and CTLA-4
Presence of HLA-DP2-CCL4/Be tetramer

control variables

PBS containing 1% FCS, 0.05% sodium azide, and 0.5 μg/mL CD16/CD32 (Tonbo; 2.4G2)
Incubation at 4°C for 30 minutes
Removal of innate cells using antibodies against CD11c, CD11b, IA/IE, HLA-DP, and Ly6G

positive controls

HLA-DP2-CCL4/Be tetramer staining

negative controls

Not explicitly mentioned

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