2.3. Immunophenotyping and function-related assays by flow cytometry
This protocol is extracted from research article:
Functional responsiveness of memory T cells from COVID-19 patients
Cell Immunol, Apr 17, 2021; DOI: 10.1016/j.cellimm.2021.104363

Immunophenotyping was performed with monoclonal antibodies anti-human-CD4 (OKT4), -CD8 (RPA-T8), -CD56 (MEM-188), -CD19 (SJ25C1), -CD45RA (HI100), -CD45RO (UCHL1), -CCR7 (G043H7), -CD25 (M-A251), -CD38 (HIT2), -4-1BB (4B4-1), -PD-1 (NAT105), -CD14 (M5E2), -CD11b (ICRF44) (BioLegend); -CD1a (REA736), -CD83 (REA714) (Miltenyi). Median fluorescence intensity (MFI) values were determined on CD4+ and CD8+ T cells, and according to CFSE dilution. The change in the MFI were calculated by comparing the data from the co-cultures with the antigen-loaded DCs and the control DCs.

The percentage of T cells with CFSE dilution was assessed for proliferation. The antigen-specific proliferation capacity of T cells was calculated as the change in proliferation wherein the data from the co-cultures with control monocyte-derived DCs were used as normalizer. The supernatants collected were used in a multiplex ELISA (LEGENDplex, BioLegend). All flow cytometric analyses were performed on a FACSAria II sorter.

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