Starting point: confluent T75 flask containing stuck GLUTag cells and 20 ml DMEM medium

All steps are undertaken in a hood!

Day 1: Splitting a confluent flask

1. Label one 50 ml falcon tube with “condition” and one with “rubbish”
2. Transfer the DMEM from the flask into the falcon labelled “condition”
3. Wash cells with 10 ml PBS and transfer the dirty PBS into the “rubbish” tube
4. Add 3 ml trypsin to the flask and leave it in the incubator for 3-5 min
5. Check under the microscope that the cells have detached
6. Add 5 ml fresh medium to stop the trypsinisation reaction
7. Spin the cells down at 600 rpm for 5 min
8. Chuck the supernatant
9. Resuspend the cells in 5 ml fresh medium and triturate them 20 times with a 5 ml stripette (Note: using a 5 ml stripette rather than a bigger-volume one will help separate the cells due to the narrower aperture of the pipette)
10. Add 5 ml condition medium from the “condition” tube to the resupsended cells
11. Add 3 ml of the resuspension to a new T75 flask containing 17 ml fresh DMEM medium
12. The other 7 ml can be used to seed Petri dishes for use in experiments, e.g.: calcium imaging, electrophysiology, secretion experiments, transfection, etc.

Day 3: Changing medium

Exchange 10 ml supernatant for 10 ml fresh DMEM medium

Day 5: Splitting a confluent flask

Refer to Day 1.

Cristian Riccio, Institute of Metabolic Science, University of Cambridge

Acknowledgments

Thanks to Dr. Edward Emery and Dr. Frank Reimann from the Institute of Metabolic Science for their contribution to the establishment of this protocol.