Flow Cytometry

Permeabilization and Fixation

Staining intracellular antigens like cytokines can be difficult because antibody-based probes cannot pass easily through the plasma membrane into the interior of the cell. In order to accomplish this, cells should first be fixed in suspension and then permeabilized before adding the antibody. The choice of fixative is an important first step. Formaldehyde and gluteraldehyde create bonds between lysine residues resulting in cross-linked proteins, however gluteraldehyde increases autofluorescence. It is usually used at concentrations of 0.5-4% in PBS depending on the sample. If you are going to store your samples for longer periods of time they should be removed from the fixative after 1-2 hours.

Formaldehyde will not permeabilize the samples so a separate permeabilization step is needed. This allows probes to access intracellular structures while leaving the morphological scatter characteristics of the cells intact. Triton, digitonin and saponin are examples of permeabilization reagents which act by disrupting the cellular membrane. The level of permeabilization is important as epitopes access may require different levels of permeabilization (e.g. cytoplasmic vs nuclear epitopes) and unbound antibody has to be sufficiently washed out of the cells. There are also many commercial kits available today that provide the reagents to carry out both fixation and permeabilization, for example, Leucoperm™ (Figure 28).

Fig. 29 Intracellular staining.

Fig. 28. Intracellular staining. Human whole blood (A) was surface stained for CD4 followed by intracellular staining for IFN- gamma, on resting lymphocytes (B) or lymphocytes stimulated with PMA/Ionomycin and treated with monensin (C) prior to staining.

Alcohols as Fixatives

Alcohols are also used as fixatives, typically as a cold 70% solution, that fix by denaturing proteins. The benefits here are that they also permeabilize the cell membrane and are suitable for long term storage at 4oC or -20oC. Epitopes can however be masked by the denaturing process with alcohol fixation, so optimization may be required. Alcohols as a fixative are most commonly used for DNA analysis.

Triton is a trademark of Dow Chemical Company.