Phospho-flow is such a cool technique. It can also be a bit tricky to get it right. The events happen quickly, so you have to be quite precise. Also, because phospho groups can be added and removed by cellular enzymes, you generally need to use different fixation and permeabilization reagents than with other intracellular staining techniques.

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Peter Krutzik's protocol from Gary Nolan's lab is very good. BD has lots of technical resources for phospho-flow, as well as specialised reagents for particular applications such as combining transcription factor and phospho-epitope staining (although I'll note that this works reasonably well using the cheaper approach below).

 

A couple improvements developed with help from Stephanie Hublet-Baron, Carly Whyte and Amy Dashwood:

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• Stain overnight. Improves sensitivity, uses less antibody. 

• Do the overnight staining at room temperature in the dark. Allows better access to the nucleus. Gives pretty good transcription factor staining.

• Do the staining in Tris-buffered saline rather than PBS (with FCS or BSA and EDTA). Reduces background.

• Use formalin rather than PFA. Preserves epitopes better.

• Use synthetic small molecule dyes (e.g., Alexa Fluors, Spark dyes, Brilliants, Reals, etc.) for anything stained prior to fixation. Prevents fluorophore degradation by methanol.

• Stain whatever you can post-fix. Reduces the chance of the antibody causing phosphorylation events through agonist signalling.

• Stain viability and any surface markers, wash, then stimulate. Eliminates wash steps at the end of the assay, allowing you to directly add the formalin to the cell culture well.

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If you prefer to use a kit, there are many reagents that BD has developed. They also have their own protocols.

Nucleotide incorporation allows us to monitor cellular proliferation both in vitro and in vivo. This might be useful for cell cycle measurements, tracking antigen-specific clonal bursts or looking at repopulation of niches. 

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Traditionally, these assays were performed using BrDU, which required denaturation of the DNA to detect the incorporated nucleotide analog with an antibody. This made it difficult to combine BrDU with other measurements. The Nobel Prize-winning Click Chemistry changed all of this, and EdU incorporation now allows for co-staining for cell surface markers, cytokines transcription factors, fluorescent proteins or phospho-proteins. The Click-It Plus EdU kit is generally the best option.

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Some tips:

• Titrate the fluorescent azide reaction as you would with an antibody. Optimal concentrations are typically much lower than kit recommendations.

• The fixation buffer provided by the kit is a pretty strong PFA solution and will cause loss of many epitopes for post-fix staining (e.g., transcription factors).  The fluorescent azide is small enough that nuclear access is not a big issue. Most kits, particularly those designed for transcription factor staining, will work well.

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A couple relevant publications:

Flomerfelt & Gress protocol.

Sun et al., early paper using EdU in vivo.

Again, a really cool technique that tells us about cell-to-cell communication. This is a functional assay in a way that many flow cytometry experiments aren't. There are many protocols out there. Here's Florian Mair's. Here's what the Liston lab does.

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The basic intracellular cytokine staining protocol looks like this:

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• Stimulate cells, typically 4hrs with PMA, ionomycin and monensin or brefeldin A.

• Perform surface staining.

• Fix

• Permeabilize

• Stain for cytokines

• Wash

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These tend to be harder experiments to fit into a 9-5 workday schedule. Here, performing overnight staining for cytokines allows you to leave earlier as well as really improving detection.

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Important things to optimize:

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• Stimulation time. 4hrs is adequate for most things with a strong polyclonal stimulus.

• Ionomycin. Ionomycin concentration is critical. Ionomycin potency varies by vendor.

• Phorbol ester. Most people use PMA. Gitta Stockinger's group introduced phorbol 12,13-dibutyrate (PdBU), which is more water soluble and less toxic.

• Secretion block. Monensin reduces viability more than Brefeldin, but may be important if you're looking at granule elements like granzyme.

• Fixative. Formalin gives great cytokine staining while not completely killing your epitopes for transcription factors and surface markers. PFA-based buffers such as BioLegend Fixation Buffer, CytoFast and BD's Cytofix can give slightly better cytokine staining, but at a cost of greater epitope loss. BD's Cytofix/Cytoperm avoids a lot of these epitope losses.

• When to stain. Many markers get downregulated or internalized, particularly with PMA. Some (e.g., CD4) can be recovered by staining after fixation and permeabilization. Since markers are also affected by the fixative, you may achieve the best resolution by staining both surface (pre-fix) and intracellular (post-fix).

• Time. Move quickly, especially post-stimulation, to prevent cell death and cytokine secretion. Secretion blockers (brefeldin) may be added to the surface staining mix.