Flow cytometry with one fluorochrome - (Jun/22/2012 )

Hi everybody!!

I'm starting work with flow cytometer (FACS Canto II) and I have some problems. I try to detect caspase 3 in macrophages upon camptothecin and E. coli contact. I use unstain non-stimulate control, FITC-conjugated non-stimulate control and my samples. Do I have to do compensation If I have one fluorochrome?? And which population should I gate??? Please give me some advice..

Anula

I haven't worked with macrophages but have done a lot of FC with immune cells. When you are using a single colour, you don't need to do compensations. When you gate cells, try SSC-A against FSC-A and gate all the cells to the right. The cells which lie near to the 0 on the axes represent the dead cells and debris. You would want to exclude them.

Thanks a lot for Your reply:)

Have You ever examined expression of receptor by using FC?

Could you elaborate on what you would like to do? Which receptors are you interested in?

Hi!

I'm a PhD student and I'm going to examine expression of PAR2 receptor on macrophages upon E. coli contact. I am a new one in Flow cytometry and I am not sure for example what controls I should include in my experiment....

Thanks for any advice:)
Have a nice day
Anula

I am assuming that you are using a macrophage cell line. In case you are not using purified macrophages, you might want to include cell markers specific for macrophages so that you can gate everything else out. Some markers used for identifying macrophages include CD11b, Ly6C and F4/80. You would have to use an antibody against PAR2 as well to look at its expression. Make sure you select different colours for the markers that you are using and that you do single stain controls. You would have to do compendation here as you are using more than one colour.

In case you are using a macrophage cell line, you could use an antibody which binds to the PAR2 receptor in a colour of your choice. You could treat some macrophages with E.coli and leave some without E.coli contact. You can then stain both the treated and untreated cells with the antibodies and see how the expression varies upon contact.

Hi!

I'm dealing with THP-1 (human monocytic cell line) that I differentiate to macrophages. So if I well understood I should use as a control only macrophages without contact with E. coli??? What about some positive control???

Anula

For positive control, you have to use something which expresses the PAR2 receptor and which can give you a strong positive signal. I have no idea what that would be. If you can find something, it is better to have both a negative and a positive control. So the tubes would be

Your single stain controls
Sample tube: Macrophages treated with E.coli
Negative Control: Macrophages left untreated
Positive Control: Something which expresses a lot of PAR2