Intracellular Flow CytometryCell biology, Diagnostics
Flow cytometry is widely used in basic and clinical research to identify, quantify and sort distinct cell phenotypes within a heterogeneous population of cells. This application, known as immunophenotyping, is commonly used to detect specific immune cell types in a complex population using fluorophore-conjugated antibodies directed against specific cell surface markers. Flow cytometry also enables the analysis of intracellular processes, such as cell signaling, using antibodies to detect target signaling proteins and their induced post-translational modifications (e.g. phosphorylation) as well as activated downstream transcription factors and regulatory proteins. Cell preparation for intracellular flow cytometry involves the fixation of cells to immobilize and preserve proteins in a transient state. It also involves the permeabilization of the cell membrane in order to allow antibodies to access their intracellular targets.
Two immunofluorescence techniques can be applied for staining (Figure 1). The indirect method uses a non-fluorescent target-specific primary antibody, which is probed by a fluorophore-conjugated secondary antibody. The second approach utilizes a single fluorophore-conjugated detection antibody specifically directed against the biomolecule of interest.
Intracellular Flow Cytometry
Here, we present a protocol that described the procedure used to routinely harvest, fix, and stain cells for flow cytometry measurement of intracellular targets. Cells are fixed and subsequently permeabilized, allowing indirect staining of intracellular molecular markers using a combination of primary/secondary antibodies. The number of cells, containing or expressing the target biomolecule, are counted on a flow cytometer, either with or without sorting depending on the actual application.
Figure 1: Direct versus indirect immunostaining. Original image from Joshi, S. & Yu, D. Basic Science Methods for Clinical Researchers, 135–150. Academic Press, 2017.
Watch the video tutorial showing step-by-step instructions for the OriGene flow cytometry protocol for staining of intracellular targets in fixed cells