Computational protocol: Downregulated IL-21 Response and T Follicular Helper Cell Exhaustion Correlate with Compromised CD8 T Cell Immunity during Chronic Toxoplasmosis

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Protocol publication

[…] Splenic single-cell suspension was prepared using a standard method from our laboratory (). Briefly, splenocytes were prepared by mechanical disruption followed by red blood cell lysis. The following antibodies were used for cells surface and intracellular staining: CD4 (GK1.5), CD8β (H35-17.2), CD11a (M17/4), CD49d (R1-2), CD44 (IM7), ICOS (7E.17G9), CXCR5 (L138D7), FAS (SA367H8), GL7 (GL7), IFNγ (XMG1.2), TNFα (MP6-XT22), CD107a (1D4B), PD-1 (RMP-1), LAG-3 (C9B7W), 2B4 (eBio244F4), Tim-3 (B8.2C12), CD45.1 (A20), CD45.2 (104), and CD11b (M1/70). Live/Dead Aqua staining (Thermo Fisher Scientific) and fluorescence-minus-one controls were systematically performed. Cell acquisition was conducted with a Cytek upgraded eight-color FACSCalibur or FACSCelesta cytometer (BD Bioscience) and data were analyzed with FlowJo software. SPICE program provided by M. Roederer (NIH, Bethesda, MD, USA) was used to compute multiple markers analysis. […]

Pipeline specifications

Software tools FlowJo, SPICE
Application Flow cytometry
Organisms Toxoplasma gondii, Mus musculus, Toxoplasma
Diseases Encephalitis, Infection, Toxoplasmosis