A Basic Guide To Flow Cytometry (3 Foundational Concepts)

Mastering foundational concepts are imperative for successfully using any technique or system.  Robert Heinlein introduced the term ‘Grok’  in his novel Stranger in a Strange Land. Ever since then it has made its way into popular culture. To Grok something is to understand it intuitively, fully. As a cytometrist, there are several key concepts that you must grok to be successful in your career. These foundational concepts are the key tools that we use day in and day out to identify and characterize our cells of interest. 

Cells

Flow cytometry measures biological processes at the whole cell level. To do this, the cells need to be of good quality and in a single cell suspension. This means that the researcher needs to know a lot about their cells such as:

1. How to make a single cell suspension?

This is easy with liquid samples. The difficulties arise when working with adherent cells or solid tissues. The Worthington Tissue Dissociation guide is a good first place to check when dealing with solid tissues 

2. How large is the average cell?

This is especially important for cell sorting, where the size of the cell dictates the size of the nozzle, which impacts everything else. 

3. What is the size range of the target cells? 

This is important when trying to find the cells on a scatter plot. Also remember size does impact the autofluorescence signal of the cells, which could impact fluorochrome choices. 

4. Do the target cells have any unusual properties?

This could be the expression of the FcR, requiring care in blocking, unusual sensitivity to temperature or ionic concentrations and more. These special characteristics are critical in dictating the experimental design. 

Fluorochromes

The ability to identify our target cells is foundational to flow cytometry. We do this by labeling cells with antibodies against our targets. To know which  antibodies are on what cells, we conjugate each antibody to a different fluorochrome. Thus, knowing the antibody/fluorochrome combination allows us to interpret the data coming off the instrument. First, it’s important to review the critical information that every cytometrist should know about the fluorochromes they are using. 

1. How bright is a given fluorochrome?

This can be determined using a variety of resources on the web or by calculating it yourself. This will give you a chart ranking the brightness of each fluorochrome. So when you ask what is bright and what is dim, it’s right there at your fingertips. 

2. What are the excitation and emission characteristics?

This is necessary to know where the fluorochrome will be measured. 

3. What is the nature of fluorochrome?

This is important as different fluorochromes have different strengths and weaknesses. For example, there are fluorochromes that are sensitive to fixation, so not the best if you must fix your cells. Likewise, is the fluorochrome a tandem dye? Using one of the many spectral viewers available on the internet is a great way to determine this (figure 1)

Figure 1: Spectra of Brilliant Violet 421™ and Brilliant Violet 605™. Notice that both dyes have a similar excitation peak around 405 nm, but BV605™ has a second excitation peak (circled in red) that is not present in BV421™. This is a good indication that BV605™ is a tandem dye. 

4. Where does the fluorochrome cause issues?

Extremely useful to know when designing a panel. The channels that it can spread into, causing a loss of resolution is important when selecting a fluorochrome. 

5. Does your fluorochrome contain a cyanide dye?

It has been shown that some cells can bind the cyanine dye independent of the antibody. This can be reduced/eliminated by proper blocking. 

Antibodies

Antibodies are the main tool we use to identify and characterize our cells of interest. There are many thousands of different clones on the market that the researcher has to choose from carefully. As Bradbury and Plückthurn discussed in their 2015 paper, as much as 50% of the money spent on antibodies is wasted. Thus the third thing the researcher must understand is this important tool.  

1. What antibody clone should be used?

 This can be found using resources such as the primary literature and sites such as Benchsci, Antybuddy, and CiteAb, to name a few.  Researchers can find out lots of information about the different antibodies and clones available, how they perform in different assays and more. 

2. How much of the target is expressed on cells?

This becomes important when designing a panel, and historically has been difficult to get good data.  Recently Kalina and coworkers, and Amir and coworkers have published papers that can help with this process. 

3. Was my antibody validated?

 This is a big question for many antibodies. If it has not, then it becomes important for the researcher to characterize the antibody themselves. With tools like CRISPR, it is possible to selectively knock out genes to show the antibody is specific to the target of interest. 

4. How well was the antibody characterized?

Clonal drift is a known phenomena, and yet many researchers accept that their antibodies derived from hybridomas are monospecific. Bradbury and coworkers published a study where they showed 31.9% (59 out of 185) of hybridomas studied had “… one or more additional productive heavy or light chains….”.  If there is concern over the antibody quality, one should consider turning to recombinant antibodies, which are made in cultures using the DNA sequence of the antibody in question.   

Concluding Remarks

In this blog, I have outlined three foundational areas that the researcher needs to understand, to Grok, to be successful in executing their flow cytometry experiments. This focused on the cells of interest, the fluorochromes and the antibodies. These three foundational areas are central to good flow cytometry and the researcher should ask themselves what they know about each, and avoid following blindly. Otherwise, one might end up discovering that the antibody being used in the field may not be specific, as Andersson and coworkers showed regarding estrogen receptor beta (ERꞵ) antibodies – where among the 13 studied, only 1 antibody was specific for ERꞵ. 

What other foundational ideas do you think the flow cytometrist should know?  Share your thoughts on our Facebook page!  

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