While being a Control Freak may not be the best choice for living in spiritual harmony, it is a downright asset in flow cytometry research! Controls not only help you set up an experiment to get a clear or “true” answer, they can also help you troubleshoot what may have gone wrong when your data just doesn’t look quite right, so that your next attempt will turn out better. In flow cytometry, controls are critical to help determine “real” events from artifacts. So, what should you use as controls in tetramer experiments?
First let’s talk about negative controls. These are important in helping you set the region or gate around antigen non-specific T cells, so that you will know where to look for positive (i.e. antigen-specific) T cells. For example, in a dot plot displaying CD8 (x-axis) and class I tetramer (y-axis), a quadrant gate would be drawn based on the sample stained with a negative tetramer, ideally. The upper right quadrant (CD8+Tetramer+) is where the antigen-specific T cells events will appear when stained with the same panel containing the antigen-specific tetramer. An appropriate negative control for a particular tetramer will have four basic elements:
Often stocked tetramer products or customs with specificities unrelated to the experiment may be appropriate, but ultimately, it is up to the investigator to select the control most appropriate for his or her particular model or experiment. Here are some tips:
In mouse tetramer experiments, there are generally two options for selecting a negative control that will allow proper region/gate placement: