at what density i should seed cardiomyocytes (isolated from neonatal mice) in a 35mm culture dish? and what should be the final volume (0.5, 1 or 2ml )? i have to use this for ROS detection using Confocal microscope.
Thankyou for your response.
i have to use h202 to cause injury to the cells ,and my drug for protection. and then during ROS protocol, i need to wash the cells almost 4 times. dont you think that this H2O2 and then Multiple washing will decrease the number of cells to be detected? because in my last try, the cell number was low and then after ROS protocol, all the cells were gone, and i couldnt see anything under the confocal microscpe.
MY PROTOCOL iS as follows
'' after incubation (24hrs) with drug and h202 (100uM) , i wash cells with DMEM, then add 10uM/L of DCFH-DA, enough to cover the cells (almost 1 ml in those dishes) , incubate for 20-30 minutes and then wash three times with DMEM. Add Dmem fourth time and visulaize under confocal."
PLEASE CHECK IF THERE IS ANY MISTAKE WITH PROTOCOL?
thank you for your suggestions , i was able to get the data using 10uM h2O2. but i was wondering if there is any protocol for 100uM , because my earlier results are based on 100uM concentration.