Cross platform validation of a unique method for deep genomic and proteomic analysis of rare immune cell populations

Abstract

Abstract Advances in flow and mass cytometry expand the number of cell parameters that can be interrogated improving our understanding of the myriad immune cell types. These technologies, however, are limited in the types of analytes profiled in a single sample. In addition to efforts to understand the immune system for the treatment of autoimmune diseases, the immune system is increasingly a target for cancer therapeutics. These efforts require deep characterization to understand rare cell populations, requiring next-generation methods that expand on flow cytometry. 3D Flow™ Analysis integrates flow cytometry cell sorting and the nCounter® Vantage 3D™ RNA:Protein Immune Cell Profiling Assay* to characterize sorted immune cell populations with direct digital counting of 770 RNA and 30 proteins. To determine if the method is agnostic to the cell sorting technology used, 3D Flow analysis was run with the Becton Dickenson FACSARIA IIu, Sony SH800z, and Beckman Coulter MoFloXDP. Stimulated and unstimulated PBMC were co-stained with flow antibodies for CD3, CD8, and CD4 plus 30 DNA-labeled antibodies for NanoString readout. 5,000 CD3+/CD4+ and CD3+/CD8+ cells were sorted on each platform and immediately lysed to release cellular RNA and DNA tags from NanoString antibodies. RNA and DNA tags were directly hybridized to NanoString barcodes and run on the nCounter system. Expression of 30 proteins and 770 RNA across cell populations correlated between the three sorting platforms, showing 3D Flow is agnostic to the sorting technology used. Importantly, high-plex RNA data was obtained without additional molecular biology methods, such as RNA purification or library construction, simplifying incorporation into a flow cytometry laboratory.