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Tips and tricks for intracellular flow cytometry
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Flow cytometry protocols and staining procedures vary depending on whether the antigen to be detected is located on the cell surface or intracellular. Detection of cell surface proteins, such as CD markers, is relatively straightforward and apart from blocking of Fc receptors the protocol requires no extra steps. However, when staining intracellular proteins such as cytokines or transcription factors, both fixation and permeabilization steps are required prior to antibody staining.
A fixation step is essential to preserve the cellular morphology. Without it the cells will lyse when permeabilized. The choice of fixative is an important first step. Formaldehyde, which creates bonds between lysine residues to cross-link proteins, is commonly used. It is used at concentrations ranging from 0.5%-4% depending on the target antigen and cell type. However, higher concentrations can lead to increased autofluorescence. Alcohols at concentrations of 70% can also be used for fixation. They work by precipitating proteins and allow long term storage at 4oC or -20oC. They have the added benefit of permeabilizing lipid membranes but the denaturing process can lead to epitope masking therefore some optimization of your fixation process may be needed.
In order for the antibody to penetrate the plasma membrane the membrane must first be permeabilized. This is usually performed using detergents, with the epitope location determining the choice of detergent to be used. Stronger detergents are required to allow staining of nuclear epitopes as both the plasma membrane and nuclear membranes have to be permeabilized. Often for nuclear staining, alcohols are used as they are stronger permeabilization reagents. We also recommend including permeabilization buffer in your washes to remove excess antibody and prevent high background levels.
For staining of intracellular antigens, Bio-Rad offers Leucoperm buffer set (BUF09), which contains fixative and permeabilization reagents that have been optimized to guarantee maximal staining without altering the cellular morphology. A list of flow cytometry tested antibodies for which we highly recommend the use of the Leucoperm buffer set, BUF09, can be found below.
Intracellular staining. Human whole blood was surface stained for CD4A647 (MCA1267A647) followed by intracellular staining for IFN- gamma FITC (MCA1554F) after treatment with Leucoperm (BUF09), on (A) resting lymphocytes or (B) lymphocytes stimulated with PMA/Ionomycin and treated with Monensin prior to staining.
In addition to carefully choosing the most suitable fixative and permeabilzation reagents, the following factors should be considered when designing intracellular flow experiments: