Flow Cytometry Dos and Don’ts for Multi-Omics Applications

In April, we hosted our first Bench Tip webinar that featured three post-doctoral fellows who shared their practical knowledge and expertise in preparing cells and reagents, setting up assays, assessing instruments and controls, sorting cells from various sample types , and more. This article is based on the talks presented during that webinar. Read on to learn more about the use of flow cytometry in diverse applications, and then we encourage you to listen to the webinar presentations and the live Q&A that followed to get even more insights on this topic.

Natural killer (NK) cells are white blood cells that play a critical role in innate and adaptive immunity. Oscar Aguilar, Ph.D., a postdoctoral fellow in the Department of Microbiology and Immunology at the University of California San Francisco School of Medicine, studies the role of NK cell-mediated immunity in cancer and viral infections. “When NK cells encounter a cancer or infectious cell, their receptors get activated and release cytotoxic granules and cytokines to kill those cells,” explained Aguliar.

NK cells have different activating or inhibitory receptors, and cytokine receptors, hence multi-parametric approaches are important for studying these cells. “I absolutely need flow cytometry for ex vivo immunophenotyping of NK cell subsets, looking at expression of the different markers on these cells.” Flow cytometry involves sample processing to generate single-cell suspensions, which are then stained with fluorophore-conjugated antibodies. As the cells pass through the lasers in the flow cytometer, the light emitted is used to analyze the different types of cells in the sample based on their size, granularity, and other parameters.

Why choose flow cytometry?

Aguilar uses flow cytometry not only for immunophenotyping, but also for in vivo adoptive transfer studies and for performing in vitro functional assays such as NK cell activation, cell proliferation, cytotoxicity, calcium influx, and more. “For adoptive transfer studies there is nothing as quick and easy as flow cytometry,” he said. “I have also considered using mass cytometry, such as CyTOF, and single-cell sequencing techniques, such as CITE-seq, but they are expensive and labor intensive, and Western blotting does not have the ability to subset cells.” Mass cytometry is essentially flow cytometry using antibodies labeled with heavy metal ion tags, instead of fluorophores. This allows more antibodies to attach to the sample, minimizing background noise and signal overlap. The time-of-flight mass spectrometry read-out helps improve precision.

Anukul Shenoy, Ph.D., a postdoctoral associate in the Pulmonary Center at Boston University School of Medicine, studies lung epithelial and CD4 T cell crosstalk in pulmonary immunity. “A major focus of my work is to find out if and how lung epithelial cells and CD4 T cells interact and how they optimize pulmonary immunity,” said Shenoy. Immunohistochemistry (IHC) and immunofluorescence (IF) can be used to stain the lung with different markers and image the epithelial and immune cells to get spatial and anatomical data of the molecular expression of markers to see how the cells interact. “However, these techniques do not provide accurate quantitative data regarding expression levels of different molecules,” added Shenoy. “They are also low throughput, subject to researchers’ bias, and a comprehensive analysis of the lung is tedious using these techniques.”

Another alternative is dissociating the lung tissue to a single-cell suspension for performing single-cell RNA sequencing. “It’s a good option for looking at the diversity of cell types present, and it offers moderate throughput,” explained Shenoy. “However, RNA expression does not always translate to the protein level. It is also not the best option for identifying T cell lineages, which need faithful readout of transcription factors that may have low RNA expression.”

Conventional flow cytometry could not offer the range of colors that he needed to identify all the different types of cells present in the lung, and mass cytometry was not a good option either, due to the loss of cells during ionization and the large batch-to-batch variability. “For our work, looking at intracellular and cell surface markers, we needed something that was high-throughput with low variability and hence we chose spectral cytometry,” Shenoy recounted.

While conventional flow cytometry uses one detector to measure the emission spectrum of each fluorophore, spectral cytometry uses all the detectors that are available. This allows for un-mixing of spectral signatures and helps resolve overlapping fluorescence spectra. However, in both techniques the anatomical and spatial interactions of cells is lost because the tissue is processed into a cell suspension. One option to overcome this limitation is combining spectral cytometry with IF, Shenoy said. Flow cytometry also tends to underestimate the absolute number of cells in an experiment as cells are lost during tissue digestion. Morphometry combined with IHC can be used to get a more accurate count.

Mikolaj Slabicki, Ph.D., a postdoctoral research fellow at the Broad Institute/Dana Farber Cancer Institute, uses flow cytometry for his functional genomics studies. He is characterizing a new class of small molecule drugs called protein degraders, which induce proximity between the ubiquitin ligase and a specific target, resulting in target depletion. “We were able to dissect the components of the drug-induced protein degradation machinery by combining flow sorting with functional genomics,” explained Slabicki. “We derived a reporter cell line with the fused target to GFP and performed a set of pooled CRISPR/Cas9 screens. Flow sorting enabled us to identify cells, which upon the knockout were resistant to drug-induced degradation. Subsequent next-generation sequencing allowed us to identify molecular machinery required for drug-induced target degradation. Flow cytometry offered a lot of flexibility and resolution for such positive selection screens.”

How to maximize the use of flow cytometry?

Knowing that flow cytometry can work for diverse applications is good, but knowing how to get the most out of it is even better. “Get familiar with your instrument,” Aguilar noted. “Find out which fluorophores are excited by the different lasers. Familiarize yourself with the excitation and emission spectra and the brightness of each fluorophore.” Know your cell biology, so you know the relative density of molecular markers expressed on the cells. Separate fluorophores that have similar spectra using different cell types and make sure the brightest fluorophores are aligned with markers that are expressed at very low levels and vice versa.

Aguilar advised using compensation beads for markers that are on rare cell populations or expressed at low levels and getting familiar with the limitations associated with the beads and how they react with certain antibody isotypes. “When setting up voltages try to maximize the signal to noise ratio and make sure your stained cell has a higher signal than the compensation bead.”

Aguilar uses two types of controls in his experiments. The instrument controls are the samples that are stimulated but not stained with the marker of interest and those are useful for setting up the gates, voltages, and the compensation matrices. Experimental controls are the samples that are unstimulated, to see what the resting NK cell looks like. “Knowing this gives you confidence in the data obtained after cell stimulation.”

Slabicki finds that dynamic range is important for his studies, and he tests different sizes of gates to find the top hits from functional screens. “Every assay needs to be optimized, but there are guidelines that are a good starting point,” he said. “A small gate identifies only very few proteins involved in degradation, whereas a big gate gives too many false positives. Somewhere in the middle there is a sweet spot where we find a high enrichment of hits involved in target degradation.” Collaborating with bioinformaticians to get the most out of the data, is also important.

According to Shenoy, it’s a good idea to run small mock experiments weeks before running the large experiment, with a good idea of knowing what to expect. He recommended testing the threshold and gain of the instrument, optimizing the tissue digestion method, identifying and titrating the appropriate amount of antibody, and using the right controls for each experiment. “Optimize and test everything,” he emphasized.

Key Considerations When Using Flow Cytometry

• Know your cell biology and the literature
• Familiarize yourself with the instrument settings, features and limitations
• Choose the right cells, cell lines, enzymes, and reagents
• Identify and titrate the right antibodies to build the best panel
• Set up the appropriate controls for your experiments
• Test and optimize the different reagents and protocols
• Run mock experiments knowing what you are expecting to see
• Repeat the experiment multiple times to gain confidence in the data
• Do diligent analysis of the data obtained