Maintain pluripotent stem cells or PSCs in six-well culture plates and confirm the cell density under a microscope. For cardiomyocyte or CM differentiation, seed PSCs into a 96-well culture plate in a PSC preparation medium. At stage one of cardiac differentiation, when PSCs reach 80 to 90%confluency, switch the medium to CM differentiation medium with two to 20 micromolar CHIR.
After 24 to 48 hours of CHIR treatment, change the medium to a fresh CM differentiation medium. At stage two or day three, replace the medium with the CM differentiation medium supplemented with five micromolar IWR1 in culture until day five. Then change the medium to the CM differentiation medium and keep it until days six to seven when the PSCs differentiate into cardiac progenitor cells, or CPC.
At day 10 or day 12, fix the cells with 4%paraformaldehyde in PBS for 15 minutes at room temperature. At the time of staining, treat the cells with a permeabilizing solution for 30 minutes at room temperature. Incubate the sample with CTNT primary antibody overnight at four degrees Celsius.
Then treat the sample with secondary antibodies in PBS containing 1%bovine serum albumin for one hour at 37 degrees Celsius. After removing the secondary antibody from the cells, stain the nuclei by Hoechst for five minutes at room temperature. Then rinse the cells three times using PBS before adding 100 microliters of PBS per well to avoid drying.
To collect brightfield and CTNT immunofluorescent images of different stages of differentiation using an automated microscope supporting live cell culture, open the software and create a new experimental design. Choose a 5X objective and a 2X2 blends for imaging. In the channels menu, add TL brightfield channel for brightfield and AF 488 and H 33 42 channels for immunofluorescence imaging.
Modify the light path in the imaging setup. In the Z stack menu, set the number of slices and intervals when scanning. In the navigation and tiles window, set up the tile regions by carrier and set 25 tiles five columns into five rows for one well.
For acquiring images, first put the cell culture plate into the sample tray and load the tray inside the microscope. If the sample consists of live cells, open the heating system and carbon dioxide pump to keep the appropriate conditions for culture at 37 degrees Celsius and 5%carbon dioxide. Open the preset experimental project.
Open the tiles menu and calibrate the position of the plate manually. In the navigation and tiles window, select the wells needed and click create to construct tile regions for these wells. Click the verify tile regions in the tiles menu and run auto focus to verify all of the wells.
Then manually correct the focus of each well. Click the start experiment button and wait for automatic imaging. Typically, it takes about 1.2 hours to scan a whole 96-well culture plate.
In the processing framework, choose image export, select the file type of the uncompressed TIFF format, or PNG format and apply.
