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Best Practices for Cytokine Analysis

Intracellular cytokine staining (ICS) is a flow cytometry method that detects the phenotype of individual cells and the cytokines they produce. This method can provide rapid multiparametric analysis of large sets of cells. The enzyme-linked immunosorbent assay (ELISA) is used to measure cytokine levels in bodily fluids.2 Cytometric bead arrays (CBAs) measure multiple proteins produced by entire cell populations simultaneously.2 In contrast, intracellular flow cytometry allows the analysis of cytokines and other inflammatory mediators produced by individual, phenotypically identified cell types within cell populations of interest.1

Cell collection and preparation

The first step of preparing the cells is cell stimulation. Adding a protein transport inhibitor during in vitro stimulation enhances cytokine detection by allowing their accumulation within the cell. The most common protein transport inhibitors are brefeldin A or monensin and should be chosen depending on the cytokines of interest.

The cells can then be incubated for a further 2 to 12 hours at 37 °C.

  Comparative analysis of different incubation periods with brefeldin A showed that periods longer than 6 hours were associated with decreased cell viability. Once the incubation is complete, cells should be harvested, stained for viability markers and cell surface markers, then fixed and permeabilised.5

Find our BD Biosciences protocol for immunofluorescent staining of intracellular cytokines and cell surface antigens.

Planning and running your multicolor experiment

You must first choose the best fluorochrome antibody combinations. There are several factors to be considered: fluorochrome brightness, antigen expression patterns and density, and antigen co-expression and spread.

To prepare the cytometer, the photomultiplier tube (PMT) voltage, compensation and gating controls must be set. Gating controls act as boundaries to establish questions in cytometry: are my cells positive or negative for the marker in question, or what proportion of my cells are positive?7

Troubleshooting for those common problems

Time parameters:
 Instability in fluidics during sample acquisition can reduce assay sensitivity and produce false population artifacts with diminished fluorescence or scatter.6 Visualisation of time versus scatter/fluorescence can control for data stability over time, meaning invalid data can be excluded.6

Dead cells:
‘Non-viable cells’ can masquerade as false positives due to non-specific binding of antibodies. Adding a live/dead cell marker can eliminate dead cells from analysis.

Presence of aggregates:
If cells stick together and pass through the laser as one cell, this can cause false positivity. Aggregates have a voltage pulse that has a lower height and larger width than that of a single positive cell, so can be identified and gated out in this way.

The Fc block:
Cells with Fc receptors can cause false positives by binding to the Fc portion of the antibody.6 Addition of BD Fc Block™ Reagent or by adding serum, such as FBS, can block this interaction.

In general, remember to check your cytometer, check your cells, and check your cytometer settings.

Learn more about intracellular flow cytometry.


Watch this video on practical strategies for intracellular flow cytometry for more information including choosing the correct protein transport inhibitor for your cytokines of interest.


  1. Freer G, Rindi L. Intracellular cytokine detection by fluorescence-activated flow cytometry: basic principles and recent advances. Methods. 2013;61(1):30-38. doi:10.1016/j.ymeth.2013.03.035
  2. Medeiros NI, Gomes JAS. Cytometric Bead Array (CBA) for Measuring Cytokine Levels in Chagas Disease Patients. Methods Mol Biol. 2019;1955:309-314. doi: 10.1007/978-1-4939-9148-8_23
  3. McKinnon KM. Flow Cytometry: An Overview. Curr Protoc Immunol. 2018;120:5.1.1-5.1.11. doi:10.1002/cpim.40
  4. Bueno C, Almeida J, Alguero MC, et al. Flow cytometric analysis of cytokine production by normal human peripheral blood dendritic cells and monocytes: comparative analysis of different stimuli, secretion-blocking agents and incubation periods. Cytometry. 2001;46(1):33-40.
  5. Adan A, Alizada G, Kiraz Y, Baran Y, Nalbant A. Flow cytometry: basic principles and applications. Crit Rev Biotechnol. 2017;37(2):163-76. doi:10.3109/07388551.2015.1128876
  6. Maciorowski Z, Chattopadhyay PK, Jain P. Basic Multicolor Flow Cytometry. Curr Protoc Immunol. 2017;117:5.4.1-5.4.38. doi:10.1002/cpim.26
  7. Maecker HT, Trotter J. Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A. 2006;69(9):1037-42. doi:10.1002/cyto.a.20333



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