Optimization of intracellular Flow Cytometry staining results with Leucoperm™
Tips and tricks for intracellular Flow Cytometry
Flow cytometry protocols and staining procedures vary depending on whether the antigen to be detected is located on the cell surface or intracellularly. Detection of cell surface proteins, such as CD markers, is relatively straightforward and apart from blocking of FcR receptors requires no extra protocol steps. However, when staining intracellular proteins, such as transcription factors, both fixation and permeabilization steps are required prior to antibody staining.
These steps are essential to preserve the cellular morphology (e.g. by fixing cells in paraformaldehyde or methanol and to ensure that the antibody is able to penetrate the plasma membrane (e.g. by permeabilization with detergents such as saponin).
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.
In addition to carefully choosing the most suitable fixative and permeabilzation reagents (depending on whether mild or strong membrane permeabilization is required; strong permeabilzation with alcohols is commonly used to detect nuclear proteins), the following factors should be considered when designing intracellular flow experiments:
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