When I treat my bacterial samples with antibiotics the autofluorescence of the cells in the fluorescence channels are changing over time. These are the same channels I use for detecting the fluorescence signals of the dyes.
I'm struggling with the compensation. Because I can't make a compensation for each treatment time-point and antibiotic concentration.
So my question is how much does the changes in autofluorescence effects the compensation? Does anyone have experience in this field or maybe a useful tip?
Thanks a lot!
What antibiotics are you using? It is known that several antibiotics can be absorb light. Perhaps bacteria is accumulation antibiotics overtime.
Hello Willemijn
I´m not familiar with autofluorescence in bacteria. Pehaps the problem is the medium or the drug used to treat the bacteria. It is important to follow the bacteria also by fluorescence or confocal microscopy in the same channels used for flow cytometry, in order to investigate if the autofluorescence is actually from bacteria not from the background. As Hannes said some antibiotic exhibit strong autofluorescence. Which dyes you use to stain the bacteria?
1- Run your unstained control (untreated) bacteria and adjust the gates.
2- It is important to include unstained samples from your antibiotic treated bacteria for each time point >> Run the samples>> look at the difference in the autofluorescence
3- Exclude the difference in autofuorescence from your positive signal after running the corresponding stained/labeled samples.
I dont really have experience working with bacteria, but in general autoflourescence itself is irrelevent for the calculation of your compensation matrix (as long as you follow the rules for preparing appropriate single-stained controls). You can find more information about this here in some landmark articles from Mario Roederer:
http://onlinelibrary.wiley.com/doi/10.1002/1097-0320(20011101)45:3%3C194::AID-CYTO1163%3E3.0.CO;2-C/abstract
http://onlinelibrary.wiley.com/doi/10.1002/cyto.a.22820/abstract
- Thanks, Hannes Luidalepp and Regina C B Q Figueiredo. Something I haven't checked.
I'm using different antibiotics, and all have a different effect on the autofluorescence. What you are suggesting Regina C B Q Fiqueiredo, I did. So from that I can say that the autofluorescence is from the bacteria, because I used different isolates as well.
I use DAPI, Ethidium Bromide and DiBAC4(3). EB and DiBAC4(3) in combination. I've seen changes in the autofluorescence for the DAPI channel and DiBAC4(3) channel.
- Thanks Mohamed Meghil, that's what I already did. Good to have a conformation!
- Thanks, Florian Mair. These articles are very very helpful! I was on the same road of thinking about the calculation of compensation. But somehow the autofluorescence causes some limitations in my research, I want to define the gaps.
Compensation will not change if your autofluorescence is changing.
Compensation will only change if you use different voltage for different time point or antibiotic concentration.
I would suggest you to not to worry about compensation. just make sure not to change voltage and when you gate for positive cells for your dye consider autofluorescence for that particular antibiotic treatment and time point.
hope it helps
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