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Flow cytometry intracellular staining protocol

General procedure describing detection of intracellular proteins in flow cytometry.

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Fixing and permeabilization:

For intracellular staining, cells can be fixed first to ensure stability of soluble antigens or antigens with a short half-life (see the special recommendations below for important exceptions). This should retain the target protein in the original cellular location.

Detection of intracellular antigens requires a cell permeabilization step prior to staining. Antibodies should be prepared in permeabilization buffer to ensure the cells remain permeable. When gating on cell populations, the light scatter profiles of the cells on the flow cytometer will change considerably after permeabilization.

N.B. Cell surface staining should be performed prior to fixation.

There are several methods available:

  1. Formaldehyde followed by detergent:

    Fixation in 0.01% formaldehyde for 10-15 min (this will stabilize proteins), followed by disruption of membrane by detergent.

    Detergents:

    Triton or NP-40 (0.1 to 1% in PBS). These will also partially dissolve the nuclear membrane and are therefore very suitable for nuclear antigen staining. It should be noted that loss of cell membrane and cytoplasm will result in decreased light scattering and also in reduced non-specific fluorescence.

    Tween 20, Saponin, Digitonin and Leucoperm are mild membrane solubilizers. Use at 0.5% v/v in PBS. These give large enough pores for antibodies to go through without dissolving plasma membrane. Suitable for antigens in the cytoplasm or the cytoplasmic face of the plasma membrane. Also suitable for soluble nuclear antigens.

  2. Formaldehyde (0.01%) followed by methanol  (SEE 3)

  3. Methanol followed by detergent:

    Add 1 ml ice cold methanol to each sample
    Mix gently. Place at -20°C for 10 min
    Centrifuge, wash twice in PBS 1% BSA

  4. Acetone fixation and permeabilization:

    Add 1 ml ice cold acetone to each sample
    Mix gently. Place at -20°C for 5 to 10 min
    Centrifuge, wash twice in PBS 1% BSA

    N.B. Polystyrene/plastic tubes are not suitable for use with acetone.

SPECIAL RECOMMENDATIONS:

Antigens close to the plasma membrane and soluble cytoplasmic antigens will require mild cell permeabilization without fixation.

Cytoskeletal, viral and some enzyme antigens usually give optimal results when fixed with acetone, alcohol or formaldehyde (high concentration).

Antigens in cytomplasmic organelles and granules will require a fixation and permeabilization method depending on the antigen. The epitope needs to remain accessible.

Intracellular staining procedure:

  1. Add 100 µl of fixative. Incubate for 10 min at required temperature (see above)
  2. Add 100 µl detergent-based permeabilizing agent and incubate in the dark at room at room temperature for 15 min
  3. Wash the cells by adding 2 ml of PBS (containing 0.1% triton or other permeabilizing detergent), centrifuge at 300 g (2000 rpm) for 5 min, discard supernatant and re-suspend the pellet in the volume remaining
  4. Follow antibody staining procedure as indicated in our ‘direct’ and ‘indirect’ protocols

Antibodies should be prepared in permeabilization buffer to ensure the cells remain permeable.

Detection of secreted proteins:

Detection of secreted proteins is difficult as the protein will be released from the cell before detection, or may degrade rapidly. Brefaldin A and other compounds are often used as a Golgi-Block. Cells are incubated with Brefaldin A which prevents proteins being released from the Golgi apparatus.  Any cells expressing the protein can then be detected.


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