How to Interpret Flow Cytometry Data
Flow cytometry is a specialized method of distinguishing between different cell types for applications, such as medical pathology. The first step of preparing the cells with visible compounds such as stains or antibodies, is straightforward. However, flow cytometry is complicated, as is the analysis. Therefore flow cytometric data should only be examined using professional tools and software such as FlowJo or CELLQuest. A thorough prior understanding of the software used, as well as the characteristics of the studied cell, are essential. Certain steps in the interpretation of flow cytometry data are universal and applicable to any software.
Things You'll Need
- Trained and licensed flow cytometry operator
- Flow cytometer
- Pre-stained or prepared cells
- Data from flow cytometry experiment
- Flow cytometry analysis software and dynamic license
Instructions
-
-
1
Import all the files from the flow cytometric experiment into the software to be used: Open each file as a density plot and set the X and Y axis for each data plot to side scatter (Y axis) versus forward scatter (X axis). Check all files to ensure they are on the same axis. Check that no cells are off the axes and that the software is not showing error messages. Note the dots, which represent the cells.
-
2
Add gates and regions (i.e. place a boundary line, circle, or square) to distinguish dead cells from live cells and large cells from small cells: Dead cells and debris are found lower on the X-Y axis. Larger cells such as blood granulocytes will also have a larger and higher side scatter profile, and depending on whether they are required in the analysis, can be gated for inclusion or exclusion.
-
-
3
Note how many cells are in each gate: This indicates the overall proportion of different cell types in the sample, and whether the flow cytometry process was successful or efficient. If cell numbers are the only result required, the analysis terminates here.
-
4
Reopen the files as histogram plots to analyze just a single parameter or characteristic of interest (such as cell cycle stage): Draw a horizontal line (a line gate) midway between the top and bottom of the peak. The software will compute the number of cells within the line that have the single unique characteristic. For example, a fluorescent cell can be detected by the flow cytometer, which then shows the quantity of fluorescent cells present in the entire sample.
-
5
Repeat step 4 but this time, examine 2 parameters by setting the X axis to one parameter (e.g. fluorescence) and the Y axis to the other (e.g. specific stain for a stage of the cell cycle): This indicates the proportion of cells that are simultaneously positive for both characteristics. For example, a green fluorescent cell blood cell can be stained red for specific blood granulocyte markers. Cells which are 'double-positive' for both fluorescence (seen as green dots) and the marker (seen as red dots), should appear as one very large or very high peak.
-
1
Tips & Warnings
Flow cytometry is a complex scientific technique and the analysis often requires extensive expertise and training. Much of what can be accurately interpreted from such data is determined by expertly trained scientists as the rates of false-positives can be prohibitively high. It is therefore recommended that the data be analyzed by several such scientists and the results agreed upon before a definitive conclusion is reached.
