Staining of intracellular antigens requires fixation followed by permeabilization, allowing antibodies to reach their target. These techniques, along with resources and products to optimize your staining, are described here.
Leucoperm™ makes intracellular antigen analysis using flow cytometry easy. The kit comes with a ‘Reagent A’ that fixes cells in suspension, and a ‘Reagent B’ that subsequently permeabilizes them.
With Leucoperm, antibodies can access intracellular structures leaving the morphological scatter characteristics of the cells intact. Leucoperm is superb for detecting many intracellular antigens including:
The specific formulation of Leucoperm reduces background staining and allows the simultaneous addition of permeabilization medium and fluorochrome-labeled antibodies.
Most commercially available monoclonal antibody conjugates can be used with Leucoperm reagents.
Bio-Rad Erythrolyse is designed for use in whole blood immunofluorescent staining procedures and is suitable for use with human, rat, and mouse blood.
Direct staining of intracellular antigens and cytokines by flow cytometry: Leucoperm method
Method for cell permeabilization required prior to intracellular staining using Leucoperm Accessory Reagent.
The detection of intracellular antigens requires a cell permeabilization step prior to staining. For the detection of cell cycle antigens such as PCNA and BrdU, methanol modification is recommended — see protocol FC8 (Direct staining of intracellular antigens by Flow Cytometry: Leucoperm plus methanol method).
The method described below produces excellent results in our hands; however, other permeabilization techniques have been published, and may also be used successfully for this application. These methods should always be used in conjunction with the product and batch specific information provided with each vial. A certain level of technical skill and immunological knowledge is required for the successful design and implementation of these techniques; these are guidelines only and may need to be adjusted for particular applications. Note: Specific methodology for blood appears in [ ] brackets.
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.
This flow cytometry guide aims to give you a basic overview of all the important aspects of flow cytometry.
With chapters on instrumentation, useful reagents, controls, experimental set-up, and much more, this guide enables best practice to be followed and gives practical advice on building multicolor panels with example protocols.
This guide is invaluable to beginners wanting to start flow cytometry and is a handy tool for teaching others about this powerful application.
To further assist learning we have recently added a flow cytometry glossary of terms to this guide.