Before placing your costly experimental sample on the instrument, it is important to realize that the scales of the dot plots and histograms that the instrument produces are completely ambiguous. As you recall from our discussion of the PMT detectors, the sensitivity of the instrument can be increased or decreased simply by raising or lowering the voltage applied to the detector. In order to draw any meaningful conclusions from flow data, the plot scales need to first be calibrated. This is done by running a negative control.
A negative control is a sample of the cells under investigation which has not been fluorescently labeled but has, in all other ways (incubations, spins, permeabilization, etc), been treated identically to the experimental sample. This sample is placed on the instrument and the detectors are adjusted (via voltage/amp change) in order to place the negative events in the first decade. The image above shows a negative control prior to and after adjustment. Notice that if a negative control calibration was not completed, the negative sample shown in the unadjusted plot would appear to be negative for PE (true) but positive for Fitc (FALSE!!!!).
Negative controls often fall into one of two categories: Unstained and Isotype.
- Unstained – An unstained sample is just what the name implies. The sample has not been treated with or exposed to any fluorescent reagents. It is sometimes referred to as an autofluorescence control as it is primarily useful for adjusting voltages and indicating the samples innate ability to fluoresce under laser excitation. (Remember: the power density of laser light is very high and everything will fluoresce to some degree, so it is important to know whether the sample fluoresces to a significant degree.)
- Isotype – An isotype control is very similar to the unstained control. The difference is that it has been “stained” with an isotype reagent. That is a reagent that has the same isotype as the target antibody (IgM, IgG, etc) as well as the same fluorochrome but with no epitope-specificity. With no epitope-specificity, it is not expected that any fluorochrome will bind to the cells. However, some cells exhibit a tendency for non-specific binding. If a cell is prone to non-specific binding of a particular isotype, the control samples will show some degree of fluorescence which will have to be considered during the final analysis. If a sample shows 4% non-specific binding, for instance, the final results of the experimental analysis can only be considered plus or minus that 4%!
While many investigators choose to run only one type of negative control, a well-designed experiment includes both unstained and isotype negative controls.
A final negative control to be considered is the Secondary Control. When using a two-step (secondary antibody) conjugation, it is important to run a secondary control. This control is an aliquot of the cells exposed to the secondary (fluorochrome-conjugated) antibody without prior staining with the primary antibody. Again, no binding of the fluorochrome-bearing reagent is expected, but as with anything, it’s generally not a perfect system. Often, there is some tendency for the secondary antibody to non-specifically bind to the cellular membrane causing a false positive signal. The extent of this binding must, again, be quantified and considered in the final analysis.
Another necessary instrumental control is the positive control. You have already seen positive controls in lesson 3. A positive control is an aliquot of the cells being studied stained with a single fluorochrome. An individual positive control sample is necessary for each fluorochrome used in the experimental sample. Therefore, a three-color T-/B-cell enumeration experiment would include three positive control samples (one for each color). The primary purpose of the primary control is to adjust compensation as indicated in the image above where the first plot shows a Fitc-only sample prior to compensation and after compensation.
Experimental Control Experimental controls are familiar to individuals involved in any sort of research. As such, we will not spend any significant time discussing them. However, be aware that it is not uncommon to include an experimental control in flow cytometry experiments in order to verify that everything is working as expected. A typical example would be a cell cycle control. That is, prior to running a multi-sample cell cycle experiment (perhaps as part of a larger apoptosis or drug sensitivity panel), it is not uncommon to run a control sample with a known cell cycle distribution between the phases. This ensures that proper staining has occurred as well as validating instrument operation.
Wrapping Up In order to asses your understanding of the material thus far, please email the answers to the following exercise to the address below. After receiving this I will provide you with access to the next module.
- I am often asked why we use the negative control to place the negative population in the first decade of the axis as opposed to further up or further down the scale. I am now passing this question on to you. From a practical standpoint, why do we fill the first decade of the plots with negative events? Post your thoughts and engage your fellow students to determine the “best” answer to this question.