Department of Pathology, New York University School of Medicine, New York, United States; LEO Foundation Skin Immunology Research Center, University of Copenhagen, Copenhagen, Denmark; Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark
Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, Farmington, United States; Department of Biostatistics and Epidemiology, School of Public Health and Health Sciences, University of Massachusetts, Amherst, United States
LEO Foundation Skin Immunology Research Center, University of Copenhagen, Copenhagen, Denmark; Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark
Simultaneous measurement of surface proteins and gene expression within single cells using oligo-conjugated antibodies offers high-resolution snapshots of complex cell populations. Signal from oligo-conjugated antibodies is quantified by high-throughput sequencing and is highly scalable and sensitive. We investigated the response of oligo-conjugated antibodies towards four variables: concentration, staining volume, cell number at staining, and tissue. We find that staining with recommended antibody concentrations causes unnecessarily high background and amount of antibody used can be drastically reduced without loss of biological information. Reducing staining volume only affects antibodies targeting abundant epitopes used at low concentrations and is counteracted by reducing cell numbers. Adjusting concentrations increases signal, lowers background, and reduces costs. Background signal can account for a major fraction of total sequencing and is primarily derived from antibodies used at high concentrations. This study provides new insight into titration response and background of oligo-conjugated antibodies and offers concrete guidelines to improve such panels.
