I am trying to measure ROS using confocal imaging. I tried to grow cells on cover slips. After 24 hours I add the stress causing agent, then after 24 hours I wash the cells with PBS. After washing the cells, I incubate the cells with DCFDA dye for 30 mins, then wash twice and fix the cells with 4% formaldehyde. After this I hardly have cells remaining on the cover slips.
Stressed cells do not attach very well to cover slips. You either have to prepare the slides, buy prepared ones or use microplates specially for cell growth AND fluorescence microscopy (black wall, clear flat! bottom with a surface for cell growth).
I would definitely prefer to harvest the cells after 24 h if possible, do the incubation with DCFDA in suspension and measure by flow cytometry. So you can work with even small amounts of cells and you can quantify it.
I don't know exactly but I went to one of Cheng's talk and I remembered that he does ROS using confocal imaging, maybe you can check some of this papers. He has full research on this type of ROS sensing.
http://www.cls.edu.cn/english/PrincipalInvestigator/pi/index1633.shtml
24hr is too long. 3hr incubation with stress causing agent should do fine. At 24hrs the cells may be dead. Also PFA fixing is not needed, it will cause non specific fluorescence. Live cells in coverslip in 24 well plate, treat for 3hrs, (at 2.5hrs add DCF-DA and incubate 30 minutes to complete 3hr), pick the coverslip and stick it to slides and image immediately.
https://www.researchgate.net/publication/299607409_Novel_PKC-z_to_p47phox_interaction_is_necessary_for_transformation_from_blebbishields
Article Novel PKC-? to p47phox interaction is necessary for transfor...
yes , i also agreed with Goodwin.. long incubation with stress causing agent like H2O2 leads to more stress and damage cells, you can optimize by adding at different time intervals.
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