Mean Fluorescent Intensity (MFI) is often used to compare expression of target of interest (TOI) across samples/ cell populations in Flow cytometry. It gives reliable information about expression/ presence of TOI within the experiment. However, more often than not we need to compare samples from different experiments (ex: a sample ran on Flow weeks/months apart). A number of factors could influence MFI of the same TOI in a similarly treated sample such as
1. Tiny differences in staining that are beyond our control (ex: cell viability)
2. Changes in laser power
3. Flow cytometry calibration
4. Differences in compensation
Is there a way we can negate these confounding factors and normalize MFI to make it comparable between experiments. (Something other than normalizing it to a control and reporting as percent control)
Please leave your expert opinions. Thanks!
Shins
Hey,
I need to compare flow data routinely across different instruments and different sites. There is a few ways to do this and you could benchmark in a number of ways. If you are doing immunological research you can use Immunotrol reagents: https://www.beckman.com/reagents/coulter-flow-cytometry/antibodies-and-kits/clinical-research-systems-and-kits/6607077
If you bought a large batch you would be able to test on a weekly /monthly basis to see if your compensation or MFI has changed for each target in a known panel.
If you're like me though and you want to have more discrete control you can use either Spherotech 8 peak rainbow beads or my favorite the Full Spectrum beads from Bangs labs https://www.bangslabs.com/sites/default/files/imce/docs/SS%20Full%20Spectrum%20Web.pdf
I use these full spectrum beads to set a value for signal to noise and an intensity that each color should come out at. The beads don't change over time so you can keep running this as a daily QC to know if your intensity is drifting at all.
Best of Luck,
Mike
This paper has an interesting and relatively simple way of doing it. Article Development of a standardized flow cytometric method to cond...
Will try this out. Is there anyone else who used this approach?
Hey,
I need to compare flow data routinely across different instruments and different sites. There is a few ways to do this and you could benchmark in a number of ways. If you are doing immunological research you can use Immunotrol reagents: https://www.beckman.com/reagents/coulter-flow-cytometry/antibodies-and-kits/clinical-research-systems-and-kits/6607077
If you bought a large batch you would be able to test on a weekly /monthly basis to see if your compensation or MFI has changed for each target in a known panel.
If you're like me though and you want to have more discrete control you can use either Spherotech 8 peak rainbow beads or my favorite the Full Spectrum beads from Bangs labs https://www.bangslabs.com/sites/default/files/imce/docs/SS%20Full%20Spectrum%20Web.pdf
I use these full spectrum beads to set a value for signal to noise and an intensity that each color should come out at. The beads don't change over time so you can keep running this as a daily QC to know if your intensity is drifting at all.
Best of Luck,
Mike
Thanks Mike! Spectrum beads seems like a highly considerable option.
Thanks
Shins
Hey Shinsmon,
I forgot to mention if you are afraid your antibodies may be drifting you can always use the AbC beads from Thermo as well that will effectively do the same job. That being said the beads in that case a IgG coated to bind mouse, hamster and rabbit IgGs so if you using a different species you may be SOL. These are also meant to do compensation for your runs and they work magnificently well especially if you don't have large samples and you can't afford to burn any on single stain controls for compensation. And bonus, they come with a negative (unstained) bead population so you can tell what the signal to noise will be at your established PMT voltage (obviously you need to titrate your reagents before that though for it to make sense).
https://www.thermofisher.com/order/catalog/product/A10497
Mike
Each time you do an experiment include the antibody capture beads.(these are usually your single colour controls for compensation). Compare the difference in MFI intensity to previous experiment. You can either adjust the MFI on the beads to previous intensity or just take note of the variation and take that into account at the end of the run. But the variation in intensity should be very small between similar experiments...large differences between runs require troubleshooting of the protocol and the instrument.
The ab capture beads are useful as they also show deterioration in the antibodies particularly in tandem-conjugates, and sample handling errors. Other fluorescent beads will allow you to control the instrument but not issues with the sample prep.
chhers
Ann
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining