Surface and Intracellular Staining Protocols for Flow Cytometry

Abstract

The goal of flow cytometry is to quantitate protein expression on cells or classify cells based on the expression of a particular protein. This technique is widespread as it is used in both basic developmental biology labs and the clinic. In either case, the cells need to be processed into a single-cell suspension for evaluating surface and intracellular gene expression. Here, we will discuss the various staining buffers that are used in flow cytometry. In addition, we have outlined detailed protocols for carrying out both surface and intracellular staining in human and rodent tissues such as whole blood and PBMCs. Specifically, for surface staining we outline the various protocols for detection of basic surface markers, chemokine receptors, biotinylated antibodies, and LAMP proteins in natural killer (NK) cells. For intracellular staining, we outline each of the protocols for the various Fixation and Permeabilization buffers systems commonly used including Formaldehyde/Saponin, the FoxP3/Transcription buffer set, and Formaldehyde/Methanol. Finally, in each section, we discuss the various protocols for staining directly conjugated versus unconjugated antibodies.