There’s a lot of information out there on the internet – some of it is good and some of it is not so good. Typically peer-reviewed publications are great, but if you’re not familiar with the field it can be a bit tricky to navigate older publications and determine if they’ve stayed relevant as technology has advanced. This blog post has two parts – first I asked the flow cytometry experts on the Purdue list to submit their favorite publications, and second are my personal picks. If you are a researcher running flow cytometry experiments – either just starting out or you have experience – these publications should help you get high quality data.

Flow Cytometry Publication Database

I sent a message to the Purdue cytometry list with the following instructions:

I’d like to crowdsource some answers from the flow cytometry experts around the world. I’m looking to compile a list of publications that are helpful for learning flow cytometry. Here’s what I’m looking for:
-Publications that are peer-reviewed, can be found on PubMed
-Provide information that about fundamentals, best practices, or general methods for flow cytometry. Ideally providing practical tips.
-Content must stand the test of time and still provide expert advice appropriate for running an experiment in 2022

In total, 30 submissions were made. Of those, there were three that were submitted by multiple people:

Cossarizza A, et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition). Eur J Immunol. 2021 Dec;51(12):2708-3145. doi: 10.1002/eji.202170126. Epub 2021 Dec 7. PMID: 34910301. This was the most popular submission, with two people submitting the third edition and one person submitting the second and first editions.

Liechti T, Weber LM, Ashhurst TM, Stanley N, Prlic M, Van Gassen S, Mair F. An updated guide for the perplexed: cytometry in the high-dimensional era. Nat Immunol. 2021 Oct;22(10):1190-1197. doi: 10.1038/s41590-021-01006-z. PMID: 34489590.

Park LM, Lannigan J, Jaimes MC. OMIP-069: Forty-Color Full Spectrum Flow Cytometry Panel for Deep Immunophenotyping of Major Cell Subsets in Human Peripheral Blood. Cytometry A. 2020 Oct;97(10):1044-1051. doi: 10.1002/cyto.a.24213. Epub 2020 Aug 31. PMID: 32830910; PMCID: PMC8132182.

Each of these two papers were submitted by two people.

Find the full list of the publications here:

Download the full database

My Picks for Best Publications

Prior to asking the Purdue list for help I compiled my own a list of publications that I find useful for running a flow cytometry experiment in 2022. Below are my selections. Note that these choices and the database above were not influenced by each other, and I did not make any submissions to the database.

Flow Cytometry Basics

Shapiro HM. Practical Flow Cytometry. Print ISBN:9780471411253 |Online ISBN:9780471722731 |DOI:10.1002/0471722731 Why you should read it: This is basically the bible of flow cytometry.

Herzenberg LA, Tung J, Moore WA, Herzenberg LA, Parks DR. Interpreting flow cytometry data: a guide for the perplexed. Nat Immunol. 2006 Jul;7(7):681-5. doi: 10.1038/ni0706-681. PMID: 16785881. Why you should read it: This is a seminal paper for flow cytometry. It’s a great overview of flow cytometry best practices – especially “Box 1 Suggested Guidelines for FACS Data Presentation”. Note: it is an older paper, so when it discusses “current common practices” those are no longer common thanks to this paper.

Roederer M. Compensation in flow cytometry. Curr Protoc Cytom. 2002 Dec;Chapter 1:Unit 1.14. doi: 10.1002/0471142956.cy0114s22. PMID: 18770762. Why you should read it: This covers everything you need to know about compensation. For a brief introduction, you can also check out the author’s website: http://www.drmr.com/compensation/

Maecker HT, Trotter J. Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A. 2006 Sep 1;69(9):1037-42. doi: 10.1002/cyto.a.20333. PMID: 16888771. Why you should read it: This publication provides a fantastic summary of gating controls. It also discusses setting voltages on PMT detectors (found on our Fortessas) and compensation controls (see my note of caution below). It’s a good reference to use to refute a reviewer asking for isotype controls.

A note of caution: The publication mentions using the same marker conjugated to different fluorophores for all compensation controls – this can be risky and add a bunch of complications to your experiment. A more updated recommendation is to ideally use the exact same antibodies and tissue for your controls and fully stained samples. If that is not feasible, find an expert to discuss your specific experiment and determine the best controls.

Hulspas R, O’Gorman MR, Wood BL, Gratama JW, Sutherland DR. Considerations for the control of background fluorescence in clinical flow cytometry. Cytometry B Clin Cytom. 2009 Nov;76(6):355-64. doi: 10.1002/cyto.b.20485. PMID: 19575390. Why you should read it: This publication provides a detailed explanation of what causes issues with the fluorescence of the negative population, controls to pinpoint the cause, and recommendations for solutions. If you’re unsure if an antibody is staining the appropriate marker or not you’ll want to read this one.

Tung JW, Heydari K, Tirouvanziam R, Sahaf B, Parks DR, Herzenberg LA, Herzenberg LA. Modern flow cytometry: a practical approach. Clin Lab Med. 2007 Sep;27(3):453-68, v. doi: 10.1016/j.cll.2007.05.001. PMID: 17658402; PMCID: PMC1994577. Why you should read it: For someone brand new to flow cytometry, this paper provides a nice overview of the steps within an entire workflow of a typical flow cytometry experiment. However, it should be noted that this paper is from 2007 and that some of the information is outdated (especially the suggested software that links to broken web addresses).

Mahnke YD, Roederer M. Optimizing a multicolor immunophenotyping assay. Clin Lab Med. 2007 Sep;27(3):469-85, v. doi: 10.1016/j.cll.2007.05.002. PMID: 17658403; PMCID: PMC2034273. Why you should read this: This publication has a lot of great practical tips on preparing a flow cytometry experiment, and goes into detail about panel design.

High Dimensional Flow Cytometry

Mair F, Tyznik AJ. High-Dimensional Immunophenotyping with Fluorescence-Based Cytometry: A Practical Guidebook. Methods Mol Biol. 2019;2032:1-29. doi: 10.1007/978-1-4939-9650-6_1. PMID: 31522410. Who should read this: Anyone running a high parameter panel.

Ferrer-Font L, Pellefigues C, Mayer JU, Small SJ, Jaimes MC, Price KM. Panel Design and Optimization for High-Dimensional Immunophenotyping Assays Using Spectral Flow Cytometry. Curr Protoc Cytom. 2020 Mar;92(1):e70. doi: 10.1002/cpcy.70. PMID: 32150355. Who should read this: Anyone designing a panel for the Cytek Aurora.

Ferrer-Font L, Small SJ, Lewer B, Pilkington KR, Johnston LK, Park LM, Lannigan J, Jaimes MC, Price KM. Panel Optimization for High-Dimensional Immunophenotyping Assays Using Full-Spectrum Flow Cytometry. Curr Protoc. 2021 Sep;1(9):e222. doi: 10.1002/cpz1.222. PMID: 34492732.  Who should read this: Anyone running a high parameter experiment. This publication provides a step-by-step guide (and video tutorials!) for optimizing your assay as well as a troubleshooting table to identify issues. Although the authors use the Cytek Aurora, many of the steps should still work for other platforms.

Liechti, T., Weber, L.M., Ashhurst, T.M. et al. An updated guide for the perplexed: cytometry in the high-dimensional era. Nat Immunol (2021).  Who should read this: Anyone running and analyzing high parameter experiments, especially those using computational analysis algorithms. This publication may also be useful for anyone trying to assess if these types of experiments were performed correctly.

Hopefully both of these approaches captured most of the publications out there. But if your favorite publication is missing, feel free to comment below!

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