UT, MD Anderson, Science Park - Research Division Flow Cytometry Controls

Controls allow the facility manager and the principle investigator to evaluate the quality of the experiment and sample processing. Labeling with a species relevant immunoglobulin of the same isotype is paramount in discriminating any non-specific binding. Flow cytometers only quantitate the differences between negative and positive controls, not absolute values with the exception of intercalating dyes or with calibrated bead standards. Multiple color flow cytometry requires single color positive controls to perform adequate compensation.

Isotype Controls: Each fluorochrome labeled antibody has a species specific source and immunoglobulin isotype, i.e. IgG-1, IgG-2a, etc.. For each color employed an Ig control sample is acquired to set the population within the first log decade. For example in discriminating Mouse T cells with Rat (IgG-2b) anti-Mouse CD3-FITC requires a population labeled with Rat IgG-2b FITC. For indirect labeling the secondary fluorochrome labeled antibody would be sufficient, i.e. Donkey anti-Rat FITC if it can be demonstrated that the primary does not cross react.

Example from Jagannadha K. Sastry, Ph.D. & Pramod N. Nehete, Ph.D.,

Department of Veterinary Sciences, M.D. Anderson Cancer Center, Bastrop Veterinary Division

Principle Investigators follow a standard operating procedure when applying Flow Cytometry to their research goals.

1) Perform a literature search. Mainly to obtain an optimized protocol for the type of analysis they wish to perform that is relevant to their cells of interest. Protocols do not necessarily work on all cell types.

2) Provide the flow cytometry personnel with a copy of any publications being referenced. This allows our service to better provide the quantitative data in the format that the Principle Investigator requests.

3) Provide proper controls. In addition to single color, positive, negative, and isotype controls providing a suitable cell source allows troubleshooting of the labeling protocol. If one is following a reference, using the identical cells and reagents as used in the publication while applying the protocol to a unique cell type will help determine whether the unique cells are conducive to a given set of analytical and experimental criteria. For example when labeling a challenging cell type for DNA content such as fibroblasts including mouse thymocytes, (which label very easily) permits one to determine if the fixation/staining protocol selected is suitable or requires further optimization.

4) Plan. First run experiments are notorious for being time consuming. If you have made an appointment please allow extra preparation time so you can be punctual. The time slot after yours may be filled.

5) Use a microscope. Correct cell density can be critical in sorting and staining protocols. In addition it will verify the integrity of your single cell preparation.

6) Be objective. Do not take bad data personally. Optimizing parameters and protocols is part of the discovery process. There is no charge for the analysis of small sets of pilot samples to check reagents, staining, controls, and prep time.

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Last updated: 20-August-02