The use of the same batch for tandem conjugates is recommended. Tandem dyes (eg PE-Cy5) are more problematic due to variation in chemical conjugation. If a marker of interest is rare or possibly absent in the control cells, a different antibody directed against a more common marker can be used as long as it carries the same fluorochrome. If this is not possible, consider the use of compensation beads.Īlternatively, different cells for your compensation controls can be used as long as they express the markers of interest. Ideally, the positive and negative populations in the control samples will be the same type of cell. The autofluorescence of the positive population, before staining, should be the same as the negative control. The positive should be at least as bright as anything that will be encountered in the experiment and should form at least 10% of the population. These controls should be solely used to set compensation. Compensation controls are required for each fluorochrome and should contain both a positive and a negative population. Some fluorochrome combinations should be avoided if possible (eg APC and PE-Cy5), given the high degree of emission overlap.Dead cells, clumps and debris should be excluded from further analysis. Adjust forward scatter and side scatter so that the cell population is clearly delineated. Set voltages for fluorescence channels using an unstained sample.Ensure that the cytometer is performing within specifications using standard beads.We always recommend reviewing the flow cytometer manufacturer's instructions for detailed compensation guidelines.
However, the following guidelines should be suitable in most cases. The procedure for setting correct fluorescence compensation is essentially the same on any cytometer but there are differences between the various available instruments, which makes it difficult to provide a "one size fits all" protocol. (PE-Cy5 + PE overlap) - (PE overlap) = accurate PE-Cy5 results General procedure
What is compensation?Īll fluorochromes have excitation and emission spectra.
Please refer to the manufacturer's instructions and software manual for a more detailed compensation procedure for your instrument.
This article describes why compensation is required for flow cytometry and how to apply it.