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Lesson 9 - Controls
Flow Cytometry
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Designing an experiment is not, of course, as easy as picking appropriate
markers and fluorochromes. In this lesson, we will examine control
samples. What do they do and why are they needed?
Instrumental Controls
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.
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davadams@umich.edu Last updated:
March 8, 2005
