Using Flow Cytometry with Epigenetics
When Epigenetics met Flow
The central dogma of DNA to RNA to proteins has become increasingly out-of-date since it was introduced in the mid-twentieth century. Mechanisms in the regulation of expression, RNA processing, and translation have revealed a more complicated picture of how proteins are made and regulated. One of the major mechanisms of gene regulation is epigenetics, the study of heritable factors that do not involve DNA base alterations. Prominent epigenetic mechanisms include DNA methylation and histone modifications that alter accessibility to transcription machinery. This field has wide-ranging applications to cancer, neuroscience, development, and more.
Applying epigenetic insights to immunology has proven difficult. The standard applications like ChIP and Western blots provide aggregate, population-level information, but immunology is driven by the study of the intricate map of cell types in the hematopoietic system. Due to its ability to analyze single cells, flow cytometry has proven to be an invaluable resource in understanding the hematopoietic system, but studying epigenetic marks in conjunction with cell markers requires careful consideration and experimental planning.
Experimental Considerations on Flow Cytometry with Epigenetic Marks
The first consideration is a conjugate with a strong signal to overcome the low abundance of epigenetic marks with respect to traditional cell surface markers. Phycoerythrin (PE) is a popular choice due to its signal strength for fluorescent-based technologies, and thulium-169 is recommended for mass cytometry applications. Another issue is the compatibility of cell surface marker antibodies with the permeabilizing agents needed for nuclear access. Methanol, for instance, can damage epitopes so all antibodies need to be double-checked before the experiment. Barcoding is also recommended to prevent doublet events. Lastly, internal controls for total histone content using a pan-histone antibody are needed for proper quantification.
Validation of Epigenetic Reagents
A challenge for epigenetic flow cytometry is finding antibodies that are not only specific for the epigenetic marks, but also are suitable for flow. The best way to test these antibodies is by using positive and negative controls with pharmacological or genetic methods. Epigenetic mark abundance is sometimes triggered by specific stimuli, such as DNA damage, which can provide an additional validation method.
Applying This Method to Immunology Studies
Epigenetic flow cytometry has already yielded interesting insights. Writing in Cell, Cheung and colleagues (PMID: 29706550) used this method to investigate aging. Prior to this study, it was unknown how aging impacted the hematopoietic system, specifically in each immune cell type’s epigenetic profile.
Using mass cytometry to analyze histone variants and modifications across more than 20 immune cell types, they found that young individuals have similar epigenetic profiles from both cell-to-cell and person-to-person standpoints. However, during aging, cell-to-cell and person-to-person heterogeneity increases. Similar results with human twin samples suggest that the variability is due to environmental effects. Additionally, immune cell types could be predicted by epigenetic profiles alone. Overall, their work demonstrates the power of combining flow cytometry with epigenetics.
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- Cheung P, Vallania F, Dvorak M, Chang SE, Schaffert S, Donato M, Rao AM, Mao R, Utz PJ, Khatri P, Kuo AJ. Single-cell epigenetics - Chromatin modification atlas unveiled by mass cytometry. Clin Immunol. 2018 Nov;196:40-48. doi: 10.1016/j.clim.2018.06.009. Epub 2018 Jun 28. PMID: 29960011; PMCID: PMC6422338.
- Cheung P, Vallania F, Warsinske HC, Donato M, Schaffert S, Chang SE, Dvorak M, Dekker CL, Davis MM, Utz PJ, Khatri P, Kuo AJ. Single-Cell Chromatin Modification Profiling Reveals Increased Epigenetic Variations with Aging. Cell. 2018 May 31;173(6):1385-1397.e14. doi: 10.1016/j.cell.2018.03.079. Epub 2018 Apr 26. PMID: 29706550; PMCID: PMC5984186.