Do you fix PBMCs before staining, and does that impact the data?

Not for our spectral flow cytometry assays, but we do fix PBMCs before staining for our mass cytometry assays and see no impact on the dynamic range of most functional markers. For our mass cytometry assays, we fix PBMCs with paraformaldehyde (PFA) before antibody staining because our panels are optimized on such samples. Hartmann et al (Cell Reports, 2019) tested how PFA-fixation before (PFA-stained) or after antibody staining (live-stained) affects mass cytometry immune profiling results. They found that immune lineage frequencies are very similar between live-stained and PFA-stained samples, with an r=0.94 correlation. Hartmann also saw that PFA-fixation does not change the dynamic range of most functional markers; however, a small subset of antigens (e.g. CCR7, CD11b) showed decreased staining. The authors concluded that PFA-fixation before antibody staining has minimal effects on immune profiling data.