Harvesting the cell (adherent cells)
- Remove the culture medium
- 1ml of PBS is added to each well.
- Scrape out the cells and transfer it to Eppendorf tube.
- Take 1 ml of culture medium (which has cell) in an Eppendorf tube.
- A short centrifuge (18-20 seconds) is done.
- The pellets are obtained in the bottom of the tube.
- Carefully vacuum out the liquid leaving the pellet behind.
- 100 ml of CCLR lysis buffer is added to the pellet
- Vortex to mix. Keep the tube in the ice to keep it cold in between successive vortex.
- After the pellets dissolve; incubate in ice for 10 minutes.
- Spin at 12000rpm for 20 seconds in 4°C.
- Keep the 96 well plate for luciferase (OPAQUE PLATE) on ice.
- 10µl of supernatant is added to each well of 96 well plate.
- Now the mixture is ready for measurement.
LUCIFERASE ACTIVITY MEASUREMENT
- Just before the measurement add 50 µl of luciferase substrate to each well.
- Measure the activity using spectrophotometer.