Use of flow cytometry for epigenetic studies

Prominent epigenetic mechanisms include DNA methylation and histone modifications that alter accessibility to the transcription machinery. This field has a wide range of applications for use in cancer research, neurosciences, developmental biology, and more.

Meeting point between Epigenetics and Flow Cytometry

The central dogma of molecular biology is based on the fact that DNA is converted to RNA and RNA is converted to proteins. However, this principle has become increasingly outdated since it was introduced in the mid-20th century. Mechanisms related to the regulation of gene expression, RNA processing and its translation have revealed a much more complex picture of how proteins are produced and regulated. One of the main mechanisms of genetic regulation is epigenetics, which studies factors that do not involve alterations of the DNA base. Prominent epigenetic mechanisms include DNA methylation and histone modifications that alter accessibility to the transcription machinery. The study of epigenetic mechanisms has a wide range of applications in cancer, neuroscience or developmental biology.

The application of epigenetic knowledge to immunology has proven complex. Standard applications such as ChIP and Western blot provide biased information about the individual behavior of each cell in a sample. Due to its ability to analyze cells individually, flow cytometry has proven to be an invaluable resource for understanding the hematopoietic system, but the study of epigenetic fingerprinting in conjunction with cell markers requires careful consideration and experimental planning.

Experimental considerations on the use of flow cytometry with epigenetic fingerprints.

The first consideration to take into account is to have a conjugate that has a strong enough signal to overcome the low abundance of epigenetic fingerprints with respect to traditional cell surface markers. In this case, phycoerythrin (PE) is a popular choice due to its signal intensity. On the other hand, thulium-169 is recommended for mass cytometry applications. Another limitation to take into account is the compatibility of cell surface marker antibodies with the necessary permeabilizing agents to gain access to the nucleus. In this case, methanol can damage epitopes, so all antibodies should be checked twice before the experiment. The use of barcodes is also recommended to avoid doublet events. Lastly, internal controls are needed for total histone content using a pan-histone antibody for proper quantitation.

Validation of reagents for epigenetics

A challenge for epigenetic flow cytometry is finding antibodies that are not only specific for assessing epigenetic marks, but are also suitable for the flow itself. The best way to test these antibodies is by using positive and negative controls using pharmacological or genetic methods. The abundance of epigenetic marks is sometimes triggered by specific stimuli, such as DNA damage, which can provide an additional validation method.

Apply this method to immunology studies

Epigenetic flow cytometry has already provided interesting insights. As published in the journal Cell, Cheung et al. (PMID: 29706550) used this method to investigate epigenetic changes on aging. Before this study, it was unknown how aging affected the hematopoietic system, specifically the epigenetic profile of each type of immune cell.

Using mass cytometry to analyze histone variants and modifications in more than 20 immune cell types, they found that young individuals have similar epigenetic profiles both from one cell to another and from one person to another. However, during aging, heterogeneity from one cell to another and from one person to another increases. Similar results with human twin samples suggest that the variability is due to environmental effects. Furthermore, immune cell types could only be predicted by epigenetic profiling. Overall, his work demonstrates the power of combining flow cytometry with epigenetics.

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