Optimized flow cytometric protocol for the detection of functional subsets of low frequency antigen-specific CD4⁺ and...

Detection of low-frequency cells using flow cytometry is challenging, as the sensitivity of the analysis is dependent on the signal-to-noise ratio, and a cell frequency of 1 in 10,000 cells is accepted as the lower limit of detection for standard flow cytometry. A solution to this problem is to pre-enrich rare cell populations using magnetic-bead conjugated antibodies targeting lineage or activation markers. For measuring vaccine or pathogen induced immune responses, this method drastically increases the signal-to-noise ratio by enriching only activated (i.e., antigen-specific) cells and excluding all other peripheral blood leukocytes from the subsequent analysis. To date, magnetic enrichment of antigen-specific cells has only been described for CD4⁺ T cells processed for surface staining. The current study significantly expands the methodology to allow detection of antigen-specific CD8⁺ T cells and analysis of cells that had been processed for intracellular staining.•The protocol described here allows magnetic enrichment of PBMCs after fixation and intracellular staining steps without increasing the non-specific background.•The protocol is adapted to automated enrichment-mode on flow cytometers.•The procedure boosts the sensitivity of the flow cytometry analysis by significantly increasing the sample size of functional antigen-specific cells without skewing the composition of the functional cells pool.