Next to the flow cytometer itself, the most important component of a flow cytometry experiment comes down to the antibodies. It is by using antibodies conjugated to fluorescent markers that we are able to identify our specific cells of interest and quantitate the amount of our target on the cell.
When I started in flow cytometry, I was immediately taken by the technology, and only later began to appreciate the importance of understanding what my reagents were and how they worked.
With the development and rise of monoclonal antibodies, each lab or group gave them a different name. This name could be the specific clone, where the antibody was harvested, or perhaps the target to which the antibody bound.
You might have attended a talk where one investigator discussed their studies on VLA-4, while a second might have discussed information obtained using Clone 9C10. These are both the same thing, but it was like the wild west out there.
This led to the development of the cluster of differentiation (or CD) nomenclature that we use today. First established in 1982 at the first Internationa ...Read More
Most flow cytometry experiments work with antibodies conjugated to a fluorochrome for some variation on immunophenotyping. However, any fluorochrome that is excited by one of the available excitation sources, and emits within the range of the detectors, can be incorporated into an experiment.
One of the great pleasures of the past was leafing through the Molecular Probes handbook, seeing what fluorescent dyes had just been released, and thinking of possible applications for them. The classic example of non-antibody directed fluorochromes are DNA-binding dyes like PI, 7-AAD, and Hoechst, but there are many others.
Dyes exist for the detection of everything from large nucleic acids to reactive oxygen species, and from lipid aggregates to small ions. Concentrations of physiologically important ions such as sodium, potassium, and calcium can be important indicators of health and disease.
Calcium ions play an especially critical role in cellular signaling. As a signaling messenger, calcium is involved in everything from muscle contractions, to cell motility, to enzyme activity.
Cells tig ...Read More
Flow cytometry is designed to measure physical and biochemical characteristics of cells and cell-like particles using fluorescence.
Fundamentally, any single-particle suspension (within a defined size range) can pass through the flow cytometer.
Beads, for better or worse, are a sine qua non for the flow cytometrist. From quality control, to standardization, to compensation, there is a bead for every job. They are important — critical, even — for flow cytometry.
Beads can do much to enhance flow cytometry, so without further ado, let’s delve into the world of beads.
1. Quality Control Beads
Starting at the top are the quality control beads. These beads represent the first line of defense for any facility to determine if their instrument is working within the specifications set by either the vendor or, more importantly, the facility itself.
Over time, each instrument’s quirks and idiosyncrasies are revealed as quality control beads are run and the data analyzed.
It is critical to make sure to analyze the trends in QC beads so that issues can be spotted before they be ...Read More
A colleague related the following conversation/mini-rant that happened in their facility just the other day. The names have been changed to protect the innocent.
New User #1: “Hi, I want to sort and run a 10-color panel on preparation of dissociated rat prostate tumor cells. Can you tell me what I need to order so we can do this next week?”
Wise Flow Director: Mental facepalm. “Can you be a bit more specific?”
New User #1: “I need to look at all the different cells in this prostate tumor and sort them for RNAseq. What colors do I need?”
Sadly, this scenario is repeated more frequently than not around the world. Let’s see how this might have played out with a more informed user.
New User #2: “Hi, I want to sort and run a 10-color panel on preparation of dissociated rat prostate tumor cells. I know what antibodies I need, but I don’t know the instrument configuration. I would like to do the sort next week.”
Wise Flow Director: “Great, here is our sorter configuration. I’m happy to help you design your panel, but next week might be a bit early to plan for the...
How do you follow best practices in flow cytometry to improve reproducibility?
Reproducibility is in the science spotlight these days. With the growing body of evidence showing how much translational research is not reproducible, funding agencies and journals are taking note.
Flow cytometry, as a technique, has changed and developed over the years, with researchers constantly evolving and evaluating best practices based on technological developments.
However, in the dark recesses of old lab notebooks, there still exist the time-worn protocols of yesteryear that come back to haunt the next generation of graduate students. The lure of getting a head start by using an already written protocol drives them to perform experiments using obsolete and outdated methods, dooming their research to the bin of irreproducible results.
It’s time to shine the light of modern cytometry on these bygone practices, and in doing so, provide tools for researchers to improve their experiments with current best practices.
1. Manual Data Compensation
In the days of analog flow cytometers, data was pro ...Read More