Cytokine staining: When to stain

Intracellular cytokine staining (ICS) is one of the more elegant yet difficult assays in flow cytometry. It reveals the functional communication between leukocytes, but the stimulation and fixation steps can make it tricky to see all the definition we want. One of the common problems is that cells will often downregulate or internalize key identifying molecules during the stimulation process, and many of these are also crosslinked by the harsh fixatives required to preserve intracellular cytokines. Standard lineage-defining markers such as CD3, CD4, CD8, CD19, NK1.1 and CD14 are all affected in one way or another.

Today we’ll look at how to deal with this issue and when is the best moment to stain for the markers.

With ICS, you have to fix and permeabilize the cells to access the cytokines. At this point, you may as well leave the sample staining overnight; you’ll almost always get better cytokine staining.

This means we have a few possibilities for when to stain for our lineage markers:

1. Prior to stimulation (cell surface)

2. During stimulation (cell surface, 37C)

3. After stimulation (cell surface, traditional approach)

4. After fixation and permeabilization (with or without overnight staining)

It’s worth mentioning that option 4 has been done before, and is in fact the recommendation from this great paper on cytokine staining. My recommendation is to either do option 4 or 3 & 4 both, but option 3 obviously works adequately for most people.

Let’s address the impact of each on workflow, viability, lineage marker detection, fluorophores and cytokine production.

Workflow

1. This adds time to your already long day or does not shorten it. For best results you will still need to stain again after stimulation with a viability marker to catch cells that have died from the stimulation.

2. This shortens your workflow. You will still need a viability stain post-stim, but this can be as quick as 10min.

3. This is the reference workflow. You’ll need one staining for markers and viability post-stim.

4. This shortens your workflow. You will still need a viability stain post-stim, but this can be as quick as 10min.

Viability

1. Reduced. The presence of azide in the antibodies is enough to cause cell death, particularly in fragile cells such as cryopreserved PBMCs. Composition of your sample may change. Certain antibodies, such as anti-CD3, anti-TCR or anti-IgM may cause signaling, activation or anergy of cells, affecting cytokine production.

2. As with 1.

3. Good.

4. Good.
   

Example data from cryopreserved PBMCs

Marker detection

1. Good marker detection. This is essentially the same as what you’d expect from fresh cells. Unless the markers are shed (like CD62L), the signal should still be present.

2. Generally good marker detection, with potential for high variability between experiments.

3. Poor marker detection, generally.

4. Good marker detection, generally.

Post-fix staining improves separation for CD3 and CD4. With this fixative (BD Cytofix-Cytoperm), CD19 and CD56 stain better pre-fix. Staining pre- and post-fix gives the best separation for all markers.

Fluorophores

1. Poor/variable. Incubating protein-based fluorophores at 37C for hours will result in loss of signal. Tandems will break down. Brilliant dyes may interact more.

2. As above, but worse. Brilliant interactions will increase, and brilliant stain buffer should probably not be used for reasons of viability.

3. Acceptable. Tandems used here will be exposed to fixative, leading to some breakdown.

4. Best option. No exposure of fluorophores to conditions that cause degradation.

Note that combining 3 & 4 is not appropriate for labile tandem dyes as the pre-fix (option 3) dye will have broken down more than the post-fix dye, resulting in spread versus the parent dye.

Cytokine production

1. Reduced when using standard azide-containing antibodies. Azide is a metabolic poison and reduces cytokine production.

2. Reduced due to addition of azide to metabolically active cells.

3. Good. Unaffected.

4. Good. Unaffected.

Exposure to azide-containing antibodies prior to or during stimulation reduces IL-2 production.

Antibodies used:

Spark PLUS UV 395 anti-CD4

BUV805 anti-CD8

PE anti-IL-2

PE/Fire-640 anti-CD19

Spark Blue 550 anti-CD3

BUV563 anti-CD56

Fixable viability dye eFluor 780

Further reading

• Lovelace & Maecker, Multiparameter Intracellular Cytokine Staining
  

  • Good tips on many aspects of the protocol

• Lamoreaux et al., Intracellular cytokine optimization and standard operating procedure
  

  • Good tips on many aspects of the protocol

• Wolfswinkel et al., Extensive flow cytometric immunophenotyping of human PBMC incorporating detection of chemokine receptors, cytokines and tetramers
  

  • Good tips on combining with tetramers

• Mair & Tosevksi. Intracellular Staining for Cytokines and Transcription Factors

• Kovacs et al., Evaluating cytokine production by flow cytometry using brefeldin A in mice

  • In vivo cytokine production. Flow could be better.

• Gong et al., An Artifact in Intracellular Cytokine Staining for Studying T Cell Responses and Its Alleviation. Useful if you work with neutrophils or tissues.