Flow cytometry is a powerful tool for analyzing cell populations based on surface and intracellular markers. Intracellular staining allows for the detection of molecules within cells, including intracellular biomarkers. Some of the intracellular biomarkers that can be examined include:
Cytokines such as IFN-γ, TNF-α, IL-2
Transcription factors (e.g., FoxP3, STAT proteins)
Signaling molecules (e.g., phosphoproteins like pERK, pAkt)
Intracellular proteins, for example, Ki-67 for proliferation and Bcl-2 for apoptosis.
Cell Stimulation for Activation of Cytokine-Related Signaling Pathways
To detect cytokines, cells are often stimulated with agents like PMA/Ionomycin or specific antigens.
Protein transport inhibitors (e.g., Brefeldin A, Monensin) prevent cytokine secretion, allowing accumulation inside the cell.
Surface Staining
Before fixation and permeabilization, surface markers (e.g., CD4, CD8) can be stained to identify cell subsets.
Fixation
Cells are fixed using formaldehyde or paraformaldehyde (PFA) to preserve cellular structure and immobilize proteins.
Permeabilization
Detergent-based buffers (e.g., saponin, Triton X-100) create pores in the membrane, allowing antibodies to access intracellular targets.
Methanol permeability may be used for nuclear targets, such as FoxP3 and Ki-67.
Intracellular Staining
Cells are incubated with fluorescently labeled antibodies against the target molecule.
Isotype controls and fluorescence minus one (FMO) controls are essential for accurate interpretation.
Immunology: Profiling cytokines in Th1/Th2/Th17 cells
Cancer Research: Detection of phosphorylated signaling proteins
Stem Cell Studies: Analysis of transcription factors such as Oct4 and Nanog.