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The X-axis is the amount of red fluorescence. The more red fluorescence a cell emits, the farther to the right the cell data will appear on the histogram. The Y-axis is the amount of blue fluorescence. The more blue fluorescence a cell emits, the cell data will appear closer to the top on the histogram. Remember, CTL's have a high level of protein B(blue) and protein R (red).
Quadrant 1 shows data for a cells with many blue fluorochromes and no red fluorochromes. Data for cells with high levels of both blue and red fluorochromes will appear in quadrant 2. If cells have neither blue nor red fluorochromes, the data will appear in quadrant 3. Data for a cell with many red fluorochromes and no blue fluorochromes attached will appear in quadrant 4.
Remember, this histogram only shows CD8+ T-cells, one type of lymphocyte (click here for a reminder on the types of T-cells). Of these types of cells, you are looking for CTLs (cytotoxic T lymphocytes). The CTLs have a large amount of Protein B (Blue fluorochromes) and low amounts of protein R (red fluorochromes). Where do they appear on the histogram?
CTLs have a high amount of Protein B on their surface, nut low amounts of Protein R. So, using the flow cytometer, you will be looking for cells that have many blue flourochromes attached with very few red flourochromes attached. You will learn how to analyze the data in the next section.
The CTLs will appear in the boxed upper left quadrant of the histogram.
|This histogram is from a nonimmune control mouse, one that was never injected with tumor cells or exposed to dioxin. Its immune system has not produced many CTL cells. The flow cytometer data tells us that the CTL count is about 6% of the CD8+ T lymphocytes.
|This histogram is from a mouse that was injected with tumor cells called the vehicle control mouse. You can see that its immune system responded by created a very large number of CTL cells. In fact, they are 72% of CD8+ T lymphocytes.
|Let’s take a look at the sample taken from the animal that was exposed to dioxin one day before it was injected with the tumor cells.
How does this mouse’s data compare to the two histograms above? Remember, you know that the mouse was injected with a tumor cell. Its CTL count is only 8% of CD8+ T lymphocytes. What is going on?