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Flow Cytometry FAQ’s

Flow Cytometry is a powerful tool used to measure … need an intro section… Flow Cytometry can present a number of unique challenges. We address some common questions about flow cytometry below. If your question is not addressed here, please feel free to submit your question and one of our scientists can contact you.

Frequently Asked Questions

  • FMO stands for Fluorescence Minus One.
  • FMO is the total fluorescence (the combined emission signal from all fluorophores that are being used) minus the fluorescence from a single fluorophore.
    • E.G., in an experiment with 5 fluorophores, an FMO sample would be comprised of 4 of the 5 fluorophores.
  • Each fluorophore used will have its own FMO (e.g. omit all fluorophores one at a time).
    • For example, in an experiment with 5 fluorophores, there would be 5 FMO samples each comprised of 4 fluorophores, omitting a different fluorophore for each sample:
      • FMO for Fluorophore #1: omit Fluorophore #1, include Fluorophores #2-5.
      • FMO for Fluorophore #2: omit Fluorophore #2, include Fluorophores #1, and #3-5 .
      • FMO’s for Fluorophores #3-5: performed in the same manner as FMO’s #1 and #2.
  • As with any proper experiment , controls (both positive and negative) are necessary to accurately interpret the data. FMO’s serve as a negative control. An FMO helps identify true baseline negative (unstained) cell populations.
  • Unstained controls can help to verify negative populations, however FMO’s are more accurate for multi-color (fluorophore) experiments.
  • There is a possibility of fluorescence spillover in multicolor experiments, where the emitted light from a different fluorophore in the sample may slightly overlap with the target fluorophore.
  • In a perfect experiment compensation would account for all fluorescent spillover, however this is not often the case.
  • FMO’s allow for the negative population of cells to be verified even in the presence of spillover from other fluorophores.
  • An FMO can be compared to an unstained sample to verify if spillover is occurring. The FMO will appear to have shifted fluorescent intensity as compared to the unstained sample.
  • This depends on the cell types and the complexity of the flow panel.
  • Without an FMO it is more difficult to precisely estimate where the cutoff (gate) between positive and negative populations should be.
    • For example, a sample may clearly indicate two distinct populations, however without an FMO it may be difficult to determine if those populations are positive/negative or if they are instead both positive (e.g. bright/dim; where bright = high expression and dim = low expression, but still positive).
  • Additionally, there may be other confounding factors such as changes in cell source: primary versus immortalized, fresh versus cultured, stimulated versus resting, and specimen (donor variability), as well as various treatments and culturing conditions which may increase variability. Having an FMO removes one source of ambiguity and provides confidence when interpreting data.
  • No, compensation beads are not a great substitute for cells. First, compensation beads do not have the same level of autofluorescence (natural fluorescence) that cells do. Second, compensation beads have a different binding profile from cells – beads will bind all the antibodies, whereas a cell will bind antibodies in different proportions. This will affect the fluorescence spillover. Last, there are certain stains that beads will not bind such as membrane dyes, DNA dyes, viability dyes, and so on.
  • The best practice is to use cells that match the test samples. If possible, FMO’s should contain both a positive and negative population. This may not always be feasible, especially when working with purified cells, or in experiments with multiple treatments.
    • In some cases, it may be beneficial to use unpurified cells, pooled cells (e.g. treated and untreated), or to perform multiple FMO’s for each part of the experiment.

 

  • Yes, the order should be matched to each sample type.
    • Example: In a phenotyping experiment for Tregs (CD3+CD4+CD25+FoxP3+), first gate on the main population of lymphocytes (FSC x SSC), then gate on singlets (FSC-H x FSC-A), next start setting FMO’s on live cells (if using viability dye), lastly set FMO for CD3, then CD4, and so on.
  • Still perform the FMO as normal in the event there is any non-specific binding that may add to potential spillover.
  • It may be useful to have a different cell type available to use as a positive control to verify that the staining protocol is working as expected.
  • If the cells are in a mixture within the same well, the FMO can use that same mixture.
  • However, if the cells are kept separate and will be analyzed separately, a separate set of FMO’s will need to be prepared for each analysis.
  • For this FMO, use cells that are maximally stimulated or matching most stimulated sample.
    • Alternatively, stimulated and unstimulated cells can be pooled together to guarantee both positive and negative populations.
  • In some cases, stimulation may affect the size, shape, and natural autofluorescence of cells. If this is a concern, a pilot study verifying FMO’s at low, medium, and high stimulation levels should be performed.
  • Cells for FMO’s should ideally undergo the exact same treatments and conditions as the test cells.
  • Isotype controls use an analogous off-target antibody, and have been falling out of use in favor of FMO’s.
    • An isotype control is an antibody of the same isotype (e.g. IgG1, IgG2a, IgG2b, IgG4) and should have the same fluorophore. The isotype will be directed against a target that is NOT in the sample, and thus functions as a negative control.
  • Isotype controls can assess the potential for non-specific binding, however there may be significant differences in binding of an isotype versus actual antibody staining. Therefore, FMO’s are preferred over isotype controls.
    • Additionally, isotype controls only work as negative controls for other antibody stains, they cannot replicate other stains that may be used (nuclear stains, viability stains, membrane dyes, etc.).
  • Treat that group of markers as a pooled stain. There should only be one FMO for a dump channel.
  • Match the staining treatments and timing to that of normal test samples – this may involve multiple rounds of staining.
  • An FMO is only as good as the choice of antibodies for the staining panel. FMO’s can help improve but will not completely fix a panel with significant non-specific binding. Make sure to test feasibility of any new panels prior to use.
  • The same rules for staining of samples apply to FMO’s – use the same stain concentration, use the same sample composition (including treatments and culture conditions), and use the same staining procedure, same gating strategy, and perform on the same instrument (ideally on the same day as the test samples: as laser intensity, PMT/CCD sensors can vary over time).
  • Take time and plan out FMO’s well beforehand.
  • Xeno Diagnostics offers expert level experience in flow cytometry services. Flow services can be paired with our in-vitro cell-based assays to support product development. Offerings include the following:
    • Phenotyping (T-cells, Tregs, NK, B-cells, monocytes, DCs, etc.)
    • Viability and Apoptosis
    • DNA replication and Cell Cycle
    • Cell division and proliferation
    • Activation markers
    • Intracellular cytokines
    • Protein and Kinase Phosphorylation/Activation
    • Receptor Occupancy
    • Efficacy and Potency Assays

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