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 coverslips.
please suggest.
How often do you check that your cells remain on the coverslip? For example, are there still cells on the coverslip after the post-treatment PBS wash? If your cells are dying, they may lose their adhesion and wash away. You might try doing your experiment in a well-plate so there is reduced fluid agitation and slowly pipette all washes in and out of the corner of the well.
The confocal microscope here does not have a state for 96 well plate, hence i tried on coverslips. yes the cells are there after i wash them with PBS. but after i load the dye and then wash, i hardly can see anything.
Is your dye packaged in DMSO? If so, are you diluting it appropriately low enough so as to preserve cell viability?
Hi Mubin, the most important thing is that you shouldn't fix your cells with PFA to analyze ROS. Ideally you should analyze live cells. However there are alternative methods for fixation and ROS analyzes with methanol and acetic acid (http://www.ncbi.nlm.nih.gov/pubmed/23178299).
Other issues are: Are your cells viable after your treatment? Which cells are you using? You could try to increase adherence with poly-L-lysine. Does your microscope have an adaptor for small petri dishes? There are petri dishes with coverslips in the middle.
Good luck!
thank you
to jordan: yes i have made stock solution of the dye using DMSO. The stock solution was made like 2 months ago and i preserve it 4 degree. On the day of experiment, i dilute it to 20 micro molar final concentration to cells.
to Fabianno
I think there is stage for culture dishes. can i grow the cells in small culture dishes and directly look at them with media or PBS in the dishes. As the laser is below and the media on cells should not effect it. Do you think its a good idea?
You should use an inverted microscope to observe cells in dishes. Remember its better to use medium without phenol red for fluorescence.
Good luck!!!
Hi, if technically feasible I would also prefer life cell analysis and avoid any fixation steps. Dependent on the ROS species you are interested in (H2O2, superoxide, hydroxyl radical, peroxynitrite etc.) a wide range of fluorescent dyes are available (e.g. from Molecular Probes, who have an excellent online selection tool) for almost any excitation laser and filter sets. DCFDA may be a good choice, but you may consider other dyes with better bleaching resistence. Please always be aware that laser excitation itself may elicit the generation of ROS, so it is advisable to image at high scanning speed and to tolerate lower resolution. If you are looking for good microchannel sytems or glass bottom dishes for imaging, you may check those supplied by ibidi. Good luck!
see this paper, you will get protocol
http://onlinelibrary.wiley.com/doi/10.1111/j.1365-3040.2011.02454.x/abstract
hello.. please help me with this.
I do ROS analysis with microplate reader as well. i load the dye to cells after 24 hrs of their growth and wash them after 30 mins, add fresh media 1% FBS and then add the nanoparticles which i m investigating if they cause ROS production. Then I take fluorescence of the plate at different time points upto 48 hours. I was curious to know if this is a good idea? does the cells have the dye in them for 48 hours and when the cells grow do the new cells also have the dye in them?