At any rate, I have a bit of time before that, so I'm going to churn out another biology lesson. It is related to what I have been working on the past three days, and unfortunately that's unrelated to what I described in my Biology 101 lesson. So, since this will be out of order, I'll just call it Biology 201. I'm sneaky like that...
Flow Cytometry
I have another project aside from the one I described here; this one is more cancer-related, and involves looking at signals that induce pre-B cell Acute Lymphocytic Leukemia (preB-ALL). To do this, I need to take tumor lymph node tissue and analyze it to determine what sorts of cells are in it, and what these cells look like. PreB-ALL affects cells that are partway through developing into B cells, one of major cell types in the immune system (ever heard of antibodies? B cells are the cells that produce antibodies). B cells, like any other cells in the blood, develop from a type of stem cell called a hematopoietic stem cell. Depending on certain signals the developing cell receives, it is directed down a path to become a specific type of cell. For example, stem cells may receive a signal to become lymphoid cells, and these cell in turn receive a signal to become B or T cells, and so on. Each stage of development has certain requirements for specific signals and for molecules that can receive and transmit these signals. That means if a cell is missing a necessary molecule, or has a mutation in the gene for that molecule, it won't be able to develop past that stage in development. There is an immune disorder, for example, in which patients are missing an important gene for B cell development, and these people therefore have no B cells; instead, they have high levels of cells that are stuck at that stage in development.
Sometimes, immature cells like this can become cancerous, as is the case with preB-ALL. The cells are stuck at a developmental stage called the pre-B cell. If there are other mutations that can act on these cells, they might develop into preB-All. I want to figure out what other signals are inducing these developmentally challenged B cells to turn into cancer. To do this, I need to look at the tumors that develop from cells that have various mutations, and determine if they are indeed preB-ALL tumors. And that brings me to today's lesson on flow cytometry.
All cells in the body can be differentiated based on special markers on their surface. Some of the most distinct cells are those of the immune system, which are covered with receptor molecules that help them do their jobs. What flow cytometry lets you do is take a mixture of cells and determine what molecules are on each one individually. It can also be used to see at what stage in development a particular cell is, because cells often display specific combinations of molecules as they go through development.
Flow cytometry takes advantage of the specificity of antibodies. Antibodies can come in a WIDE variety of flavors, but they all recognize a specific protein sequence. For example, somewhere floating through your blood is an antibody for the influenza virus. When you get a flu shot, your body is enticed to start producing copious amounts of the antibody so that you can recognize it quickly again in the future. Antibodies have the potential to recognize any protein sequence in the world, which means some can recognize molecules on your own cells. Antibodies that find their partner protein will bind to it with considerable affinity. So if you have a mixture of CD4 and CD8 T cells (which express the molecules CD4 and CD8, respectively), and you add to that mixture anti-CD4 and anti-CD8 antibodies, the anti-CD4 will only attach to the molecules on the CD4 T cells, and the anti-CD8 will only attach to the CD8 T cells.
This is where flow cytometry comes in: companies have been able to glue onto the end of antibodies fluorescent molecules. These glow a certain color when hit with a laser. When the antibodies bind to their specific molecules, it essentially attaches a fluorescent mark to that cell. So, if you have anti-CD4 antibodies that glow green, and anti-CD8 antibodies that glow red, you can determine which cell is which. A flow cytometer shoots the cells individually through a stream of liquid which passes through the lasers. A detector in the machine then records the intensity of the light the cells give off. This way, you can determine not only what molecules are on cell, but also how many. You can also have mixtures of antibodies on one cell. Some CD4 T cells are also express CD25 (CD4+CD25+). These are functionally very different cells from those that only express CD4 (CD4+CD25-). CD4+CD25+ cells suppress immune responses while CD4+CD25- cells induce immune responses. Now, if you have a mixture of CD4 and CD25 T cells, along with the same antibodies I mentioned before, and you add anti-CD25 that glows blue, you can identify each individual cell by its color combination:
Red = CD8 T cells
Green = CD4+CD25- T cells
Green & Blue = CD4+CD25+ T cells.
Pretty cool, huh? The flow cytometer can measure millions of cells individually, and you can plot the relative fluorescence on a graph and estimate how many cells are in one population versus another, like this:
As you can see, on the Y axis is CD4 fluorescence, and on the X axis is CD8. Each single dot is a cell, plotted based on its expression of each fluorescent molecule. Therefore, in this lymph node sample, 38.4% of the cells are CD4 T cells (they are CD4+ and CD8-). Likewise, 34.2% of the cells are CD8 T cells.25.9% of the cells express neither CD4 nor CD8. Within these cells are my B cells I'm looking for. Because the cytometer measures all the colors given off by each cell, we can select only that population in the bottom-left and look at what other colors they express. Like this:
So this graph represents only those cells that are CD4-CD8-.
In my sample, I added not only anti-CD4 and CD8, but also anti-B220 and IgM. These are B cell markers, and now I can look at what sort of expression is found on the CD4-CD8- population. B cells express B220 early during development and continue to express it throughout the cell's life. In contrast, IgM is only expressed on mature B cells. So, based on the graph, we can determine how many cells are developing B cells, and how many are mature B cells. If this was a tumor sample, I could use this information (combined with other antibodies) to determine what sort of cancer it is.
You can even take flow cytometry further, by using a machine that separates cells based on their fluorescence profile. This way, you can take a tissue sample and easily separate it into tubes of immature vs mature B cells, CD4+CD25+ vs. CD4+CD25- T cells, and so on.
So this graph represents only those cells that are CD4-CD8-.
In my sample, I added not only anti-CD4 and CD8, but also anti-B220 and IgM. These are B cell markers, and now I can look at what sort of expression is found on the CD4-CD8- population. B cells express B220 early during development and continue to express it throughout the cell's life. In contrast, IgM is only expressed on mature B cells. So, based on the graph, we can determine how many cells are developing B cells, and how many are mature B cells. If this was a tumor sample, I could use this information (combined with other antibodies) to determine what sort of cancer it is.
You can even take flow cytometry further, by using a machine that separates cells based on their fluorescence profile. This way, you can take a tissue sample and easily separate it into tubes of immature vs mature B cells, CD4+CD25+ vs. CD4+CD25- T cells, and so on.
