10x Genomics and BioLegend co-hosted a webinar led by Felix Nettersheim, MD, postdoctoral researcher in the group of Professor Klaus Ley at La Jolla Institute for Immunology, San Diego. Dr. Nettersheim’s studies focus on understanding how metabolic reprogramming of immune cells affects atherosclerosis, the major underlying pathology of cardiovascular disease. Single-cell RNA-sequencing (scRNA-seq) has emerged as a powerful tool to characterize immune cell populations. Yet, mRNA levels correlate poorly with expression of surface proteins, which are well established in defining immune cell types. CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) utilizes oligonucleotide-labeled antibodies to simultaneously analyze surface phenotypes and transcriptomes in the same cells, helping researchers overcome this issue.
This study was designed to (1) validate the staining of BioLegend TotalSeq™ antibodies on human peripheral blood mononuclear cells (PBMCs) and (2) confirm the optimal concentration for staining. We tested a custom lyophilized cocktail of antibodies at four different concentrations on human PBMCs, which are a commonly interrogated cell population in immunology. For most antibodies, the recommended concentration (1x) was optimal and enabled the detection of most surface proteins. For some antibodies, doubling the concentration increased the signal. A few antibodies worked even when the concentration was lower than recommended (to 1/5 or 1/25). Our data are a resource for building an informative and cost-effective panel of TotalSeq™ antibodies, which can then be used at their optimal concentrations in future CITE-seq experiments.