I had a problem with flow cytometry. I did a cultive with PBMCs in RPMI 10% Bovine Fetal serum, 1% glutamine, 1% streptomicine/peniciline, for 24 hours, 37ªC, 5% CO2. Then I stimulated the cells with PMA/ionomicine for 3 hours. I did the superficie mark and then the intracelular mark with Foxp3 staining buffer set. The negative control tube had fluorescence in APC- channel. Someone can tell me which can be the problem?
Hi,
most probably as you already suspected what you have observed here is autofluorescence, a common phenomenon caused by excitation / emission of intrinsic cellular compounds. Second, non-specific binding of your labelled antibody may also be the issue here, or an incorrect compensation, causing leakage from other channels. Finally, if you work at extreme gains / detector voltages, it may also be a minor fraction of scattered excitation light (SSC) leaking through the filters (which are never 100% effective), which is however is rather unlikely in the (far) red spectrum, but this depends on the particular optical configuration in your cytometer.
However, if you have set up your cytometer (compensation!) correctly, intrinsic autofluorescence should not critically interfere with analysis if you are dealing with medium to highly expressed markers. If you have to detect rare markers, it would be the simplest solution to just switch to a different fluorophore for the particular marker, measuring it at a wavelength where autofluorescence is weaker or absent.
Thank you very much for your answer. It is very helpful. I has been already checked the issues you proposes. In more that one opportunity I had problems with that channel of fluorescence. I will do another experiment to check if that autofluorescence influence my particular mixture of antibodies.
Best regards
Paola
Hi Paola,
How do you define your negative control? What colour is your Foxp-3 antibody on?
1) Is you negative control untreated PBMCs with surface stain and foxp-3 intracellular staining? If so, you should be able to detect Tregs in untreated PBMCs.
2) If your Foxp-3 is on APC, I will introduce a Fluorescent-Minus One (FMO) control to gate on your true positive population.
Also based on your laser configurations, there are certain channels that can bleed into APC channel namely eg. Pe-Cy5 etc. You might want to look at your laser configuration for this matter.
Best Wishes
Niva
Thank you for asking this question because I am facing the same problem. After I isolate cells from milk, I measure 3 markers using FITC, PE and APC. The FL-1 channel shows a uniform amount of auto fluorescence across all events (debris, epithelium, lymphocytes, neutrophils) while the 585-42 channel is almost completely clean. What bothers me is the specific auto fluorescence within the 660-10 channel that seems to predominantly radiate from my lymphocyte population. I am not familiar with auto fluorescence in this channel so I wonder what could cause this.
We also got this problem with autofluorescence in the APC channel with our HL-60 cells. It seems that for both our ATRA-treated and for untreated cells, a portion of the population is autofluorescent in APC. This bugs my mind since the undifferentiated cells should at least in theory be uniform... This is a problem for us since we want to use Cypher5e as an internalization marker...
If you managed to solve/figure out this issue please let us know how you did it!
- Badges
- Science topic