What goes wrong with flow cytometry?

What goes wrong with flow cytometry?

No signal/weak fluorescence intensity. High fluorescence intensity. High background/high percentage of positive cells. Two or more cell populations observed when there should be one.

How do you describe a flow cytometry graph?

Flow cytometry data is typically represented in one of two ways: histograms, which measure or compare only a single parameter, and dot-plots which compare 2 or 3 parameters simultaneously on a two- or three-dimensional scatter-plot.

Can I use FITC and PE?

Relative contribution. In some experiments FITC may be combined with other dyes, for example PE, that emit yellow and orange photons. In those cases the relative contribution of each fluorophore to the signal in a given detector must be determined (Figure 11).

Why is compensation important in flow cytometry?

Because the compensation matrix is more relevant to cytometry data — it tells us what to subtract from the detector in order to see the true fluorescence from its primary fluorophore — it is what is displayed in cytometry acquisition and analysis software.

How do I know if my flow cytometry data is bad?

How to Identify Problems with Flow Cytometry Experiment Design – Bad Data Part 3

  1. Experiment lacks single stain controls.
  2. Only compensation beads run for single stained controls (no single stained cells).
  3. Use of isotype controls instead of FMOs.
  4. Unlabeled parameters and/or tubes.

How can I improve my flow cytometry results?

Optimize permeabilization and fixation to improve detection For further improvement, increase the formaldehyde concentration gradually up to 4% or try ethanol or methanol and using other detergents including Triton X-100, or saponin without fixation or alcohol fixation.

Is APC far-red?

Allophycocyanin (APC) is an intensely bright phycobiliprotein isolated from red algae that exhibits far-red fluorescence with high quantum yields. It is excited by laser lines at 594 and 633 nm, with an absorbance maximum at 650 nm and a fluorescence emission peak at 660 nm.

Is APC brighter than PE?

Compared to PE, APC is not as”bright”. Compared to other red fluorophores such as Cy5, however, APC is still significantly brighter and an excellent choice for use in flow cytometry.

Can I use FITC and PE together?

What does compensation mean in flow cytometry?

The term “compensation,” as it applies to flow cytometric analysis, refers to the process of correcting for fluorescence spillover, i.e., removing the signal of any given fluorochrome from all detectors except the one devoted to measuring that dye.

What are the best practices in flow cytometry for polychromatic flow?

For polychromatic flow cytometry, best practices in flow cytometry is to use the automated compensation methodologies. This will ensure consistent and accurate compensation, if some rules are followed.

Is polychromatic flow cytometry error prone?

It is error prone and subject to the researcher’s judgement, unless statistics are invoked. For polychromatic flow cytometry, best practices in flow cytometry is to use the automated compensation methodologies. This will ensure consistent and accurate compensation, if some rules are followed.

How can I improve the performance of my flow cytometer?

We always recommend reviewing the flow cytometer manufacturer’s instructions for detailed compensation guidelines. Ensure that the cytometer is performing within specifications using standard beads. Set voltages for fluorescence channels using an unstained sample.