Because of the abundance of Mtb antigens already present in granulomas and involved lymph nodes (Gideon et al., 2015 (link)), these samples were not further stimulated with Mtb peptides (Gideon et al., 2015 (link)). Due to low number of cells, uninvolved lung lobes were also not stimulated. All samples were incubated in the presence of Brefeldin A (GolgiPlug, BD Biosciences) at 37°C/5% CO2 for 3.5-4 hours prior to staining. Cells were stained with a viability marker (LIVE/DEAD fixable blue dead cell stain kit, Invitrogen) and surface and intracellular markers. Surface markers include CD3 (clone SP34-2, BD Pharmingen), CD4 (Clone L200, BD Horizon), CD8 (Clone SK1, BD Biosciences) and CD20 (Clone 2H7, eBioscience).
Protocol detail
Characterization of Mtb-specific T cells
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Corresponding Organization :
Other organizations : Massachusetts Institute of Technology, Broad Institute, Ragon Institute of MGH, MIT and Harvard, University of Pittsburgh, Harvard University, Children's Hospital of Pittsburgh
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Variable analysis
independent variables
- None explicitly mentioned
- Expression of cell surface markers (CD3, CD4, CD8, CD20)
- Presence of Brefeldin A (GolgiPlug, BD Biosciences)
- Incubation of samples at 37°C/5% CO2 for 3.5-4 hours prior to staining
- Positive control: Not specified
- Negative control: Not specified
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