To begin, resuspend the skeletal muscle cells isolated from Notexin-injured mice labeled with iododeoxyuridine in one milliliter of cold serum free DMEM and count them using a hemocytometer or a cell counter. Under a fume hood, add cisplatin stock to achieve a final concentration of 25 micromolar. Vortex the tube for 10 seconds and incubate at room temperature for one minute.
Quench the reaction with ice cold DMEM containing 10%FBS and place it on ice. After pelleting and resuspending the cells, filter the suspension through a 35 micrometer cell strainer. Under a fume hood, add filtered paraformaldehyde stock solution to fix the cell suspension and vortex for 30 seconds.
Wash it two times with two milliliters of cell staining medium or CSM by centrifugation. After the final wash, aspirate the supernatant to approximately 60 microliters and thoroughly resuspend the pellet. Add 40 microliters of 2.5 times surface antibody staining mix prepared in CSM and incubate for one hour while vortexing them every 20 minutes.
After washing the sample two times with CSM, aspirate the supernatant and flick the pellet. To permeabilize the cells, under a fume hood, add 0.5 milliliters of ice cold methanol dropwise while vortexing. Wash the cells twice with one milliliter of CSM by centrifugation.
After the last wash, aspirate the supernatant to approximately 60 microliters and resuspend the pellet thoroughly. Incubate the cells with 40 microliters of intracellular antibody staining mix for one hour, while vortexing the samples every 20 minutes. After washing the cells twice with one milliliter CSM, resuspend the samples in 0.5 milliliters of the intercalator iridium solution and vortex.
