Flow Cytometry Intra-cellular Staining Optimization
Permeabilization and Fixation Method Tips
- For the staining of secreted proteins like cytokines, a protein transport inhibitor such as Monensin or Brefeldin A should be added prior to fixation/permeabilization
in order to trap the cytokines inside.
- For combined surface and intracellular staining, it is advisable to first stain for the surface markers and then fix/permeabilize as the latter can alter some
antigen epitopes and affect antibody binding.
- Fixation/permeabilization reagents alter the scatter properties as well as the auto-fluorescence of cells. Therefore it is recommended to include an unstained
control that has been treated with the same reagents.
- Binding of antibody to surface antigen can stimulate the cells and alter the expression of intracellular signalling proteins. Therefore, surface staining
should always be performed after the stimulation.
- For phosflow staining, the cells should be fixed and permeabilized immediately after the stimulation as phosphorylation is a transitory phase and quickly pass.
- Choosing the right permeabilizing agent is extremely important – one can choose between detergents like saponin or TritonX and methanol.
- Saponin does not alter the surface antigen epitopes so surface staining can be done afterwards.
- TritonX and Tween should be avoided as they can lyse cells if incubated for long.
- Methanol is compatible with most intracellular antigens and cells treated with methanol can be stored at -20 to -80°C for an extended duration without
loss of signal.
- Not all fluorochromes however can withstand methanol treatment. The table below shows which commonly used dyes are methanol resistant and methanol sensitive.
|Methanol Sensitive||Methanol Resistant|
|Alexa Fluor 647||All tandem dyes|
|Alexa Fluor 488|
Keywords: FACS staining protocol, intracellular staining procedure, perm and
fix protocol, flow cytometry antibody staining procedure, FACS antibody, flow