A user's guide to multicolor flow cytometry panels for comprehensive immune profiling

Staffan Holmberg-Thyden, Kirsten Grønbæk, Anne Ortved Gang, Daniel El Fassi, Sine Reker Hadrup*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Multicolor flow cytometry is an essential tool for studying the immune system in health and disease, allowing users to extract longitudinal multiparametric data from patient samples. The process is complicated by substantial variation in performance between each flow cytometry instrument, and analytical errors are therefore common. Here, we present an approach to overcome such limitations by applying a systematic workflow for pairing colors to markers optimized for the equipment intended to run the experiments. The workflow is exemplified by the design of four comprehensive flow cytometry panels for patients with hematological cancer. Methods for quality control, titration of antibodies, compensation, and staining of cells for obtaining optimal results are also addressed. Finally, to handle the large amounts of data generated by multicolor flow cytometry, unsupervised clustering techniques are used to identify significant subpopulations not detected by conventional sequential gating.

Original languageEnglish
Article number114210
JournalAnalytical Biochemistry
Number of pages13
Publication statusPublished - 2021

Bibliographical note

Funding Information:
Financial support to this study was provided by the Danish Cancer Society , grant no. R72-A4531 and R146-A9531-16-S2 , and Herlev-Gentofte hospital research grant .


  • Flow cytometry
  • Immune monitoring
  • Immunology
  • Myelodysplastic syndrome
  • Unsupervised clustering


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