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Flow Cytometry (and Mass Cytometry)
Flow Cytometry (and Mass Cytometry)
Even though my forum activity has massively dwindled, I figured I'd start up a discussion on flow cytometry. The intent is to discuss flow with other "flow'ers," to teach aspects of flow cytometry to those who are unfamiliar with it, and to discuss new happenings in the field of flow cytometry.
To those interested in it, flow cytometry is an incredibly useful technique used in both research and clinical medicine. The basic idea is that a sample (usually cells of some sort - human cells, mouse cells, bacteria, etc.) is "stained" with fluorescence-conjugated antibodies. The flow cytometer is a machine that passes cells in a very thin stream through a laser (or series of lasers) that excite the fluorophore on the antibody; the cytometer can then measure the level of excitation.
Here's an example of how we might use it clinically. Suppose I have a patient's blood sample, and I want to find out the composition of some of the white blood cell populations. I mix the cells with an antibody against CD4 (T-helper cells) that is conjugated to a "green" fluorophore, an antibody against CD8 (cytotoxic T-cells) conjugated to a "blue" fluorophore, an antibody against CD20 (B-cells) that is conjugated to a "red" fluorophore, and an antibody against CD64 (macrophage) conjugated to a "yellow" fluorophore. We run the cells through the flow cytometer, and can see how many cells were tagged with which antibody.
Here's an example of how we might use it in research (and perhaps some day clinically). Suppose we want to analyze a cell signaling pathway in cytotoxic T-cells, and see whether a certain transcription factor is being expressed. We stain the cells with an antibody against CD8 that is conjugated to a "green" fluorophore (the colors can be changed and swapped around); we then kill the cells with a mixture that punctures holes in the cells' membranes. We then use antibodies against the transcription factor of interest, conjugated to a "red" fluorophore. When we analyze the data, we look at cells that expressed the green fluorophore, and the we see whether those cells expressed the red fluorophore (and how strongly it was expressed).
In addition to data analysis, these principles can be used to separate cells from a mixture. Because the cells are placed into a stream of droplets, it is possible to create "sorting streams" by placing different charges on the droplet and passing them between two electrical plates. Some potential uses for this in research would be if you want to put only one type of cell through a certain type of assay that isn't flow cytometry. Clinically, some experimental therapies against cancer involve incubating a patient's dendritic cells with some of the tumor cells, and then injecting them back into the patient; cell sorting could be used to sort out the dendritic cells from a patient's blood. (I'm not sure that cell sorting is used in that case or if they use a different method; either way, it's an example.)
Very neat, right? That's a very basic explanation of it; if anyone wants to know more about flow, ask it here and then private message me to bring my attention back to this forum.
A newer technology called mass cytometry is now making the rounds. One of the problems with traditional, fluorescence-based cytometry is that we're limited in colors, and some of the colors bleed into other color channels. Mass cytometry marries a mass spectrometer to the fluidics behind flow cytometry. Instead of fluorophores, antibodies are conjugated to rare earth metals. The mass spectrometer can then detect those rare earth metals. The benefit is that there should be no problems with color emissions bleeding into each other; the downside (at the moment) is that mass cytometry eviscerates the cells that it analyzes. Thus, while it could act in place of standard flow cytometry, it can't be used as a sorter.
Lastly, some interesting news: Sony is apparently going to enter the flow cytometry business by producing flow cytometers. Yes, that's the same Sony behind blu-ray and Playstation. It doesn't seem to fit with their business strategy at all, so it's a bit puzzling... as far as I know, they have yet to release any products.
Doc, I just got nosebleeds just from the explaining...but seriously its interesting....
Wow, sounds really interesting. It must be good to get you into a lab during the summer!
What kind of work do you do in the lab, Ledgem?
I found some cool videos for
The lab I'm with for the summer is a lupus research lab. Specifically, we're doing research on biomarkers for detection of the disease (and disease state). It's pretty interesting, and may relate to why lupus patients have higher incidences of certain conditions compared to individuals without lupus.
It's a bit of a change for me, though. It's much more clinically focused than what I did in graduate school, but it's also much more focused. My previous research was about control of the entire immune system, and how to direct it against cancer (or other conditions). This feels like it's directed for the aid of a much smaller group of people. It's not a
thing, of course (and I've met a number of lupus patients in the process, which was quite interesting and gave me some greater motivation). Just different.
I'm at an institution where my wife is doing her residency, and their research group is in the process of some major restructuring. Given my immunology (and flow cytometry) background, they have me doing a variety of things. Most of my effort thus far has been on setting up one of their cell sorters... for some reason they bought one of the most expensive, most complicated cell sorters on the market, and they don't have anyone who knows how to operate it. It's a real shame. I've gotten most of the quirks down and will now begin using it, and teaching its usage (and flow) to others in the research group so that the machine doesn't go into disuse once I leave.
I'll be taking Step 1 next summer (the first of three licensing exams for practice in America; Step 1 is regarded as the hardest), and sometimes I wonder if I should be studying for it instead of doing research
Then again, most of my peers are off vacationing in exotic places and aren't studying, either, so... I guess I shouldn't feel guilty about it.
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