Staining of intracellular proteins
Flow cytometry is applicable in analysis of various intracellular molecules including phosphorylated signaling proteins and cytokines. There are to critical steps in intracellular staining protocols. In order to stain intracellular antigens, the cells need to be fixed in suspension and then permeabilized before addition of the detection antibody. This fixation/permeabilization procedure allows the antibody to pass through the plasma membrane into the cell interior, while maintaining the morphological features.
Carrying on surface and intracellular staining in the same sample, it is advisable that the staining of cell surface antigens be performed first since fixation and permeabilization steps may decrease the availability of surface antigens. The exceptions to this order of events is when you are looking at things that are time sensitive, such as phosphospecific staining after treatment.
Note: Some surface antibodies work fine on fixed cells, other do not work at all. It can be find out only experimentally.
Detection of proteins in different cellular compartments
To avoid degradation of secreted proteins, use Brefaldin A or other compounds that prevent protein release from the Golgi apparatus. Such treatment should enable the detection of secreted proteins within the cells.
Antigens close to the plasma membrane and soluble cytoplasmic antigens require mild cell permeabilization without fixation.
Cytoskeletal, viral and some enzyme antigens usually give optimal results when fixed with a high concentration of acetone or alcohol, alternatively formaldehyde.
Detection of antigens in cytoplasmic organelles and granules require individual adjustments of a fixation and permeabilization method depending on the antigen. Most of all, the epitope needs to remain accessible.
Several methods are available for cell fixation and permeabilization:
1. Fixation reagents:
1% - 4% formaldehyde (alternatively 0,05% glutaraldehyde) for 10-15 minutes in room temperature, followed by washing/blocking in PBS/2%FBS
ice-cold methanol (alternatively ice-cold acetone) - after gentle mixing place samples at -20 for 10 min, then centrifuge and wash cells in PBS/1%BSA
formaldehyde followed by methanol
Triton or NP-40 (0.1–1% in PBS) partially dissolve the nuclear membrane, that’s why these reagents are suitable for nuclear antigen staining.
Tween 20, Saponin, Digitonin and Leucoperm (0.5% v/v in PBS) enable antibodies to go through pores without dissolving plasma membrane. They are recommended for antigens in the cytoplasm or the cytoplasmic face of the plasma membrane and soluble nuclear antigens. Note: Since saponin-mediated cell permeabilization is a reversible process, it is relevant to keep the cells in the permeabilization buffer during intracellular staining. This buffer may contain also blocking agent like 2% FBS or 1% BSA
Note: Ready to use Fixation, Fixation/Permeabilization or Permeabilization/Wash buffers or staining kits are provided by vendors of flow cytometry solutions, like BD, Abcam, R&D Systems and others.
3. Further steps of intracellular staining:
A. Fc-blocking with blocking IgG (1 µg IgG/106 cells) for 15 minutes at room temperature
B. Incubation with conjugated antibody (5-10 µL/106 cells or a previously titrated amount) for 30 minutes at room temperature in the dark
C. Washing 2 - 3 times
Note: If an unconjugated primary antibody is used, incubation with an appropriate secondary antibody should occur now.
D. Cytometric analysis of cells resuspended in 200 - 300 µL PBS buffer
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