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Tandem Fluorochrome Conjugated Antibody Best Practices

Please Note: When using tandem antibody conjugates in multicolor staining panels, it is important to use exactly the same tandem conjugate for compensation tubes that are used for staining experiment samples.

Commonly used tandem fluorochromes used for flow cytometry such as PerCP-Cy5.5, PerCP-eFluor710, PE-Cy7, APC-Cy7, etc. vary in their ability to transfer energy from donor dye to acceptor dye across antibody lots and over time due to fluorescence resonance energy transfer differences. These variations result in leakage of donor dye fluorescence (e.g., PE fluorescence leaks from PE-Cy7) and diminished fluorescence emission strength (brightness) of the acceptor dye over time (e.g., Cy7 has reduced brightness as PE-Cy7 ages due to less energy transfer from PE).

When using tandem antibody conjugates in multicolor staining panels, it is important to use exactly the same tandem conjugate for compensation tubes that are used for staining experiment samples. Otherwise, compensation will not be calculated correctly leading to erroneous measurements and uninterpretable data. Tandem conjugates are also degraded by fixation making it important to run fixed samples stained with tandem conjugates as soon as possible.

Examples where the compensation tube does not equal the experiment tube:

  • PE-Cy7 (BD) ≠ PE-Cy7 (Biolegend) for any antibody conjugate
  • CD4 PE-Cy7 (BD) ≠ CD8 PE-Cy7 (BD) for compensation
  • CD4 PE-Cy7 (BD lot X) ≠ CD4 PE-Cy7 (BD lot Y)
  • CD4 PE-Cy7 (BD lot X) ≠ CD4 PE-Cy7 (BD lot X) formaldehyde fixed
  • CD4 PE-Cy7 (BD lot X newly purchased) ≠ CD4 PE-Cy7 (BD lot X one month old)
  • Substitute compensation beads for cells when the antigen density is low or the positive cells represent a low percentage of the population.

A common problem arises when choosing antibody conjugates for compensation tubes when the cell’s antigen of interest is either low density (i.e., does not yield bright staining) or the positive cells represent a low percentage of the population. This problem is exacerbated when using tandem conjugated antibodies since another antibody with the same tandem fluorochrome cannot be substituted. In these cases, instrument manufacturers and antibody vendors recommend substituting compensation beads for cells. The compensation beads can be stained with the same antibody conjugate used for experiment samples providing a brightly stained sample for compensation. This solves the problem of matching compensation samples with experiment samples when using tandem conjugates. Several vendors sell compensation beads.

  • DiVa software will accommodate multiple, same-tandem compensation tubes.

If it is necessary to use different antibodies conjugated with the same tandem conjugate for different samples in the same experiment, BD’s DiVa software accommodates this by allowing multiple compensation tubes of the same color (e.g., CD3 PE-Cy7, CD19 PE-Cy7).