Flow cytometry with one fluorochrome
Posted 21 June 2012 - 10:47 PM
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..
Posted 04 July 2012 - 11:09 AM
Posted 24 July 2012 - 04:02 AM
Have You ever examined expression of receptor by using FC?
Posted 24 July 2012 - 05:02 AM
Posted 27 July 2012 - 04:18 AM
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
Posted 27 July 2012 - 05:32 AM
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.
Edited by zodiac1505, 27 July 2012 - 05:33 AM.
Posted 27 July 2012 - 10:05 AM
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???
Posted 27 July 2012 - 10:55 AM
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