I want to know if there is a protocol to do this assay in INS-1 cell line and if mine is correct.

Day 1

1) Seed (150,000 cells) in 24 well plate.

2) Leave in the incubator overnight to allow cells to adhere.

Day 2

3) Media starved (1% FCS) for 6 hours.

4) Remove supernatant and wash 1x with warm PBS.

5) Treat cells for 30 minutes with your desired treatments (no glucose, no FCS, no phenol red) Include duplicates for each sample as one will be labelled (with 2-NBDG).

8) Remove supernatant from cells without disrupting cells.

9) Wash 2x with warm PBS and discard supernatant.

10) Add solution of 2-NBDG to half the samples (except the unlabelled controls).

11) Incubate for another 30 mins in the dark at 37 °C.

12) Remove supernatant without disrupting cells and discard solution.

13) Wash 2x with warm PBS.

14) Trypsinise cells (250 µL) for 5 mins at 37 °C and inactivate the trypsin with normal media (250 µL) transfer to Eppendorf’s (labelled with No.) and put on Ice.

15) Spin down cells in a microcentrifuge 2500 xg for 5 min or till form palate. discard supernatant.

16) Wash cells in warm PBS again and resuspend in (100 µL PBS + 1 µM of PI) to exclude dead cells which are auto-fluorescent.