Extensive flow cytometric immunophenotyping of human PBMC incorporating detection of chemokine receptors, cytokines and tetramers

Abstract

Characterization of immune cells is essential to advance our understanding of immunology and flow cytometry is an important tool in this context. Addressing both cellular phenotype and antigen-specific functional responses of the same cells is valuable to achieve a more integrated understanding of immune cell behavior and maximizes information obtained from precious samples. Until recently, panel size was limiting, resulting in panels generally focused on either deep immunophenotyping or functional readouts. Ongoing developments in the field of (spectral) flow cytometry have made panels of 30 + markers more accessible, opening up possibilities for advanced integrated analyses. Here, we optimized immune phenotyping by co-detection of markers covering chemokine receptors, cytokines and specific T cell/peptide tetramer interaction using a 32-color panel. Such panels enable integrated analysis of cellular phenotypes and markers assessing the quality of immune responses and will contribute to our understanding of the immune system.

BCG revaccination in adults enhances pro-inflammatory markers of trained immunity along with anti-inflammatory pathways

“…A minimum of 1x10 6 rested cells were incubated with cell viability dye in PBS, washed in PBS/0.1% BSA (Sigma, Merck Life Science NV, Amsterdam, the Netherlands) followed by blocking Fc receptors with 5% human serum in PBS for 10 minutes, washed and incubated with an antibody cocktail for surface markers at 4°C for 30 minutes as described previously. 81 Cells were washed twice and fixed for 10 minutes at room temp with 1% paraformaldehyde (Pharmacy LUMC, Leiden, The Netherlands). Cells were washed once more and stored at 4°C for acquisition on a 5L Cytek®Aurora (Cytek Biosciences, Fremont, CA, USA) the following day the LUMC flow core facility ( https://www.lumc.nl/research/facilities/fcf/ ).…”