Multiparametric Flow Cytometry for Granuloma Characterization
Granulomas harvested from other Mtb infected NHPs were used in the flow cytometry analysis and processed as previously published (Gideon et al., 2015 (link)). Cells were counted and stained for viability using fixable viability dye (Zombie NIR, BioLegend) and other surface and intracellular markers using the standard protocols. Surface markers include: CD3 (SP34-2, BD), CD4 (L200, BD), CD8a (RPA-T8, BD), CD8b (2ST8.5H7, BD), TCR γδ (5A6.E9, Invitrogen), CD16 (3G8, BD), NKG2A (Z199, Beckman Coulter) and intracellular markers include: Granzyme B (GB11, BD), Granzyme A (CB9, BD) and Granzyme K (G3H69, BD). Samples were acquired on a Cytek Aurora spectral cytometer (5 laser configuration) and unmixed using SpectroFlo software (Cytek). Final analysis was performed in FlowJo (v10, FlowJo)
Other organizations :
Africa Health Research Institute, Massachusetts General Hospital, University of KwaZulu-Natal, Children's Hospital of Pittsburgh, University College London, University of Massachusetts Chan Medical School
Expression of intracellular markers: Granzyme B, Granzyme A, Granzyme K
control variables
Standard protocols for cell staining and processing
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