• Comparisons
  • Inconsistent Results

Miscellaneous

In addition to low signal or high background, there may be other difficulties researchers experience when performing flow cytometry. View these miscellaneous categories to the left.

Cross Reactivity

Have you checked the compatibility of the product with your sample? An antibody that is known to react against mouse may not perform similarly with rat samples. A literature search could reveal if the clone’s cross-reactivity has been investigated. In addition, a homology comparison on Uniprot may give you a starting point to determine possible cross-reactivity. It should be noted, however, that even if an antigen displayed high homology between two species, this does not guarantee a cross-reactivity. BioLegend’s antibody cross-reactivity chart may also be a useful tool for you.

UniProt
Cross-Reactivity Chart

Fluorophores

Do you find that the PE format of an antibody from a competitor work better than the FITC format you obtained from BioLegend? Comparisons between fluors can be misleading, as the brightness between FITC and PE are quite different (on a scale of 1-5, 5 being the brightness, PE rates a 5 and FITC rates a 3). If you are looking at products between companies, you should compare the same fluors to get an accurate comparison.

In addition to differences in fluorophore brightness, there can also be differences in optimal usage amount, which means that the concentrations between products may also be different, potentially contributing to less than optimal results. Furthermore, tandem dyes can potentially have distinctly different properties among different antibody manufacturers, in terms of brightness and signal-to-noise, so be aware when switching your source of tandem antibodies.

BioLegend Fluorophore Brightness Index

Clones

In addition to fluor differences, you should determine whether the clones in the comparisons you are making are the same. Different clones recognize different epitopes and have varying binding affinities. Therefore, a direct comparison between two different clones may not be accurate.

Lot Changes

If your results have changed over time, are you still using the same lot? If you have recently changed to a new lot, you may want to check its concentration to ensure you are still using the same amount of antibody. In addition, some test size lots have changed from a recommended usage of 20 µL/test to 5 µL/test. You may also want to compare the two lots side by side to confirm the new trend is lot-dependent.

Concentration Lookup Tool

Human Variation

Are you testing on human patients/samples? While mice can be genetically identical, humans, of course, present a lot more variation from person to person. You may need to increase your sample size to draw a conclusion for your data. In addition, the disease state/health, age, and ethnicity can all affect the expression levels of certain markers. Different batches of cell lines can also produce inconsistent results. Side-by-side comparisons should always be made with the same sample/cell types.