Spectral Flow Cytometry

Experimental Methods

Spectral flow cytometry data was aquired using a five laser Cytek Aurora spectral flow cytometer, and was performed by dividing and staining PBMC samples with four separate panels of antibodies. Where possible, both scRNA-seq and flow cytometry were performed on aliquots from the same sample on the same day. As a standard practice, instrument setup and tracking is performed daily using SpectroFlo QC beads and the Daily QC program.

To enable spectral unmixing and compensation matrix analyses, we utilized healthy resting PBMCs for markers with positive signal in at least 5% of control PBMCs, and Ultra-Comp control compensation particles (Thermo Fisher Scientific) for markers with low frequency detection.

The flow cytometry panels used in our studies, including suggested gating, detailed panel composition information, and additional methodological details, are available in our paper in Cytometry Part A:

Details about the combined scRNA-seq and spectral flow cytometry pipeline are available in our paper in STAR Protocols:

The composition of the flow cytometry staining panels is provided below.

Data Processing

After acquisition of raw data for each panel on a five laser Cytek Aurora spectral flow cytometer, data were unmixed using Cytek SpectroFlo software with the ordinary least squares method. Autofluorescence extraction was not performed, as minimal signal improvement was observed in initial testing.

Antibody Staining Panels

Full reagent details, including vendors, catalog numbers, and RRIDs, are available as a supplement to Heubeck, et al. (2021), which can be downloaded here:
Download Reagent Details