What factors should I consider when designing intracellular flow experiments?

When designing intracellular flow experiments, you must first complete both fixation and permeabilization steps prior to antibody staining. 

• Fixation: Fixation helps preserve cellular morphology and prevents cell lysis during permeabilization. Choosing the appropriate fixative and optimizing your fixation process is crucial. Formaldehyde, which is a commonly-used fixative, cross-links proteins but can lead to increased autofluorescence at higher concentrations. Alcohols, which can also be used for fixation at concentrations of 70%, precipitate proteins and can also permeabilize lipid membranes, but may mask epitopes.
• Permeabilization: Permeabilization of the plasma membrane allows antibodies to penetrate and bind to intracellular epitopes. Detergents are commonly used, with the choice depending on the location of the epitope. Stronger detergents are required to stain nuclear epitopes. Alcohols are stronger permeabilization agents and are often used for nuclear staining.  

Other factors to consider when designing intracellular flow experiments:

• The staining procedure you use will depend on whether the intracellular protein to be detected is a transcription factor, phosphorylated protein, or cytokine. 
• Stain surface markers first before fixation and permeabilization to avoid potential interference.
• For secreted proteins like cytokines, use protein transport inhibitors before fixation to trap cytokines inside cells for intracellular staining.
• Your choice of fluorophore can be influenced by the staining protocol and fixative used. Larger fluorophores may not reach all epitopes and can be affected by alcohol fixation.
• Use specific transcription factor buffers when detecting surface markers and transcription factors simultaneously to avoid adverse effects on surface marker staining.
• Phosphorylation is transient, so it’s important to fix and permeabilize cells immediately after stimulation to detect phosphorylated proteins.
• A fixation or permeabilization step may decrease the brightness of tandem dyes. If your intracellular flow cytometry panel consists of tandem dye conjugated bodies, make sure your fixation and permeabilization steps are as mild as possible. Also, keep the duration as short as possible to maintain brightness.