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Flow Cytometry! OR: what I do during the day!

February 11, 2011

A cell glowing with fluorescent markers

 

I work at UCLA in the flow cytometry core facility. What that means is that I am in a room all day, surrounded by humming machines. These machines are flow cytometers. They look like this:

FACScan-- your basic, one laser, three parameter cytometer

In fact, that is a picture of one of the machines I work with. We are a core facility, which means that scientists from labs all around UCLA (and some from other institutions located in LA) prepare experiments and bring us tubes to run on the machine. These tubes have the particles they want to analyze, usually cells. I help them get set up and make sure that they get good data from their samples. I may also help them analzye the data. Either way, hopefully, they will walk away with some data they can use to cure cancer! fight HIV! make flying people! and other cool science.

Seriously though, I really enjoy talking to these researchers and finding out what questions they are asking. I’ve helped bioengineers who are designing new drug delivery particles, maybe to push past that trick blood-brain barrier and route out disease  hiding in the spaces between glial cells. I’ve assisted cancer researchers who are in the middle of a clinical trial for a cancer vaccine that can hopefully rouse a patient’ s own cells to take up arms and attack the cancerous interloper in their midst. I’ve adjusted the settings for a PI who is looking for a way to diagnose Alzhiemer disease before neurons start dying. In a tiny way, I am helping SCIENCE.

Flow– we are measuring particles suspended in a fluid stream, and cytometry– cyto means cell. Flow cytometry is a technology  that measures multiple physical characteristics of particles in a fluid stream– usually cells or other biological particles. We look at their relative size and internal complexity as well as fluorescence intensity. All of these characteristics are determined by how the cell scatters laser light and emits fluorescence in response to that light.

An LSRII from Becton Dickinson

The machines nowadays fit on the bench top, the smallest about 2 ½ feet by 1 ½ by 1 1/2 (the machine above) and the largest looking uncannily like a futuristic coffin for people five feet tall.

So I stick a sample tube on the machine and it is sucked up and surrounded by sheath fluid– a salty buffer that keeps cells moving through the machine. Optimally, the particles line up in single file and pass by the laser one by one, so we can see what each individual cell looks like.

When the cell deflects the laser light, detectors pick up the scattered light and we can see a picture of the cell populations in the sample. For example if you run a sample of white blood cells it will look like this:

The Forward Scatter is along the X-axis and dots that represent cells with larger sizes are farther along the x-axis. Side Scatter, the y-axis, is a measure of internal complexity, so round, relatively uncomplicated cells will have low ssc and cells with a lot of stuff in them and ruffled edges will have high ssc. Based on these physical properties alone, we can get some information about relative percentages of cell populations within a heterogeneous sample. But it gets way more exciting when we start looking at colors—the fluorescence of the cells.

Fluorescence occurs naturally in nature—there are algae that give off a red-orange glow and even our own blood cells glow brightly without being asked. The first fluorescent molecules that biologists used were Fluorescein isothiocyanate (FITC) a synthetic molecule that glows green and phycoerythrin (PE) a protein that makes the red-orange glow in algae.

The first cell sorters were developed in Los Alamos (dudes were looking at the effect of radiation on chromosomes) and Stanford (dudes doing immunology research). At first flow cytometers just read FITC, then PE, then a third compound called Texas Red, but the chemistry is much better now and a bunch of companies make crazy colors like  Qdots, efluors, APC, PE hooked up with all sorts of molecules, a rainbow of fluorescence. All these colors can be hooked up with antibodies to tag more than 300 different kinds of immunological markers, and many more proteins that make up cell signaling pathways or stud the membranes of cancer cells. Flow cytometry is a powerful tool. It has to be one of the only tools that scientists actually identify themselves by, donning the moniker ‘flow cytometrist’. You don’t see a lot of people walking around calling themselves ‘microscopists’ or ‘Polymerase Chain Reactionists’.

I’m just a technician, a flipper of switches, but this stuff is pretty cool.

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2 Comments leave one →
  1. Mum permalink
    February 12, 2011 12:32 pm

    Thank you for the wonderful explanation. Now I can explain to friends who ask a little better what you are doing. Knowing a better explanation may take away a little of my fun giving the example of flow cytometry being used to sex semen. GAsp! She said “sex” and “semen”. Sigh, non-science people.

  2. roese permalink*
    February 12, 2011 1:56 pm

    heehee. she said ‘science’!

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