Culturing of hESCs for Mitochondrial Analysis

1. Undifferentiated hESC HSF-6 cells are cultured on irradiated-y CF-1 murine embryonic fibroblasts.

2. Once hECSs have reached confluence, they are disassociated with either 2 mL of trypsin/EDTA (for 100-mm dish or 0.5 mL for six-well dish) or collagenase IV.

3. Cells are classified as undifferentiated or migratory for subsequent analysis. Migratory cells are those that have begun to migrate from the central colony. Differentiated cells are classified to their appropriate cell type according to relevant markers of differentiation.

3.1.1. Preparation of Cover Slips for hESC Plating

1. Heat the 0.1% gelatin solution in the 37°C water bath. Place one cover slip and add 1 mL gelatin to each well of six-well dish. Incubate overnight at 37°C to allow the gelatin to polymerize.

2. Remove the remaining liquid and return the dishes to the incubator until the coated cover slips are dry (1 h).

3. Alternative protocol:

Place the cover slips in 0.1% gelatin overnight.

Place one cover slip in each well of a six-well dish and return the dish to the incubator until the coated cover slips became dry (1 h).

3.1.2. Plate Stem Cells on Cover Slips for Immunocytochemistry

1. Change the medium in the 100-mm tissue culture dish 2 h before trypsinization and return to the incubator. Remove the medium from the tissue culture dish and wash once or twice with PBS (12 mL or with 2 mL for each six-well dish).

2. Add 2 mL of trypsin/EDTA to the tissue culture dish (for 100-mm or 0.5 mL for six-well dish) and incubate at 37°C until the colonies become detached (check the dish every 2 min under a microscope).

3. Transfer the trypsin cell suspension to a sterile conical tube and incubate at 37°C for a few more minutes, pipet the cell suspension up and down with a micropipet to dissociate the cells.

4. Add several milliliters of stem cell media, spin the suspension at low speed and remove the supernatant to ensure that the cells are no longer exposed to trypsin.

5. Resuspend the cells with several milliliters of media and split this volume in the gelatin-coated wells prepared the day before. The number of cells per well depends on how long the cells will be cultured before immunocytochemistry is carried out.

6. Make up the media volume to 2 mL and return the dishes to the incubator.

7. Feed the cells every day until they reach the optimal density for immunocyto-chemistry.

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