Flow Cytometry Intra-Cellular Staining Optimization Guide

Permeabilization and Fixation Method Tips

When to Use Protein Transport Inhibitors

For the staining of secreted proteins like cytokines, a protein transport inhibitor (e.g., Monensin or Brefeldin A) before fixation and permeabilization in order to trap the cytokines inside, ensuring their detection during intracellular staining.

Best Practices for Combined Surface & Intracellular Staining

When staining for both surface and intracellular antigens:

• 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.
• Use staining buffers compatible with both surface and intracellular antibodies to maintain signal quality.

Fixation & Permeabilization Controls for Accurate Results

Fixation and permeabilization can change the scatter properties as well as the auto-fluorescence levels of cells. Therefore it is recommended to include an unstained control that has been treated with the same fixation/permeabilization treatment to correctly adjust instrument settings and gating.

Preventing Antibody-Induced Cell Stimulation

Some antibodies binding to surface antigen can stimulate the cells and alter the expression of intracellular signalling proteins. To avoid this, surface staining should always be performed after the stimulation steps in your experimental protocol.

Phospho-Flow Staining: Fixation Timing Considerations

For phospho-flow staining, the cells should be fixed and permeabilized immediately after the stimulation as phosphorylation is a transitory phase and can quickly revert. Rapid processing preserves the phosphorylation signal for accurate detection.

Choosing the Right Permeabilizing Agent

Choosing the right permeabilizing agent is extremely important – one can choose between detergents like saponin or TritonX and methanol.

1. Saponin does not alter the surface antigen epitopes so surface staining can be done afterwards.
2. TritonX and Tween should be avoided as they can lyse cells if incubated for long.
3. 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.
4. Not all fluorochromes however can withstand methanol treatment. The table below shows which commonly used dyes are methanol resistant and methanol sensitive.

Methanol Sensitivity & Fluorochrome Compatibility

Methanol Sensitive Fluorochromes	Methanol Resistant Fluorochromes
FITC	PE
eFluor 450	APC
eFluor 660
Alexa Fluor 647
Alexa Fluor 488
PerCP
All tandem dyes

Keywords: FACS staining protocol, intracellular staining procedure, perm and fix protocol, flow cytometry antibody staining procedure, FACS antibody, flow validated

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