Highly Multiplexed Single-Cell Westerns for Exploring Heterogeneity in Protein Kinase C (PKC) Signaling and CRISPR Knockdown Efficiency

Sponsored by: ProteinSimple

Speakers

Noemi Kedei, MD

Staff Scientist, Collaborative Protein Technology Resource, Office of Science and Technology Resource, Center for Cancer Research, National Institutes of Health
BIOGRAPHY
Kelly Gardner, Ph.D.

Director of Marketing, Milo
BIOGRAPHY

Event Date & Time

DATE: April 9, 2019

TIME: 9:00 AM PDT

Abstract

In this webinar, we will provide a general introduction to the Single-Cell Western Technology (SCW) and present data from two ongoing projects from the Collaborative Protein Technology Resource (CPTR) at the National Cancer Institute. The SCW technology enables western-based detection of multiple proteins in thousands of single cells thus providing sample heterogeneity information and allowing measurement of correlations between levels and/or activation states of different proteins in a single cell.

In the first project, the heterogeneity of the Protein Kinase C (PKC) signaling pathway in LNCaP prostate cancer cell line was evaluated at baseline and after treatment with PKC activator PMA. We successfully demonstrated a highly multiplexed assay detecting 11 proteins per cell with 7 rounds of stripping and re-probing. As part of assay validation, staining specificity and reproducibility as well as stripping efficiency were assessed, and multiple reference proteins/loading controls were used and compared. Novel findings revealed by SCW include 1) identification of the expression heterogeneity of the lower MW, the higher MW or both cFos bands after PMA treatment and 2) identification of two subgroups of cells at baseline: majority of cells showing no activation and about 30% of cells with partially elevated phosphorylation signals.

In the second project, SCW was used to evaluate the efficiency of CRISPR Cas9 knockdown of NOX-1 (NADPH oxidase 1) in the LS-513 colon cancer cell line in order to facilitate clonal selection of knockdown cells. SCW identified 1) high level of heterogeneity of NOX1 expression in the parental cell line; 2) fully knocked-down clones to be used in later studies; and 3) mixed clonal populations for which traditional western blotting detected reduced NOX1 expression but missed the critical information that about 85% of cells express very low NOX1 levels and about 15% of cells express high level of NOX1.

These examples demonstrate the power of SCW to reveal single cell level information that is not detectable by traditional western blotting performed on bulk cell population.

Learning objectives:

Learn how Single-Cell Westerns help you uncover new insights about your samples including measuring heterogeneity of novel states
Learn how highly multiplexed Single-Cell Westerns are being used to study heterogeneity in signaling pathways to identify cell subpopulations with distinct activation states
Learn how Single-Cell Westerns are being used to measure CRISPR Cas9 knockdown efficiency to facilitate clonal selection of knockdown cells.

LabRoots is approved as a provider of continuing education programs in the clinical laboratory sciences by the ASCLS P.A.C.E. ® Program. By attending this webinar, you can earn 1 Continuing Education credit once you have viewed the webinar in its entirety.