Stimulation Protocol

Cell Stimulation Protocol

Initially spin down cells, lyse for RNA extraction, RT-PCR for 20, 25, 30 cycles and do serial dilution of the RNA to avoid saturation (dilue: 50x, 100x, 25x, 500x).

1. For one 90-100% confluent T-75 flask

2. Wash with 10 mL PBS

3. Trypsinize with 1.5 mL trypsin

4. Incubate for 15 minutes at 37 degrees

5.Stop trypsinization by adding 20 mL media to the flask

6. Transfer all to a 50 mL tube

7. Wash flask with 10 mL media

8. Add this 10 mL to the same 50 mL tube

9. Spin 10 minutes at half speed

10. Aspirate media

11. Add 3 mL fresh media

12. Pipet up and down with a 3 mL syringe and a 19.5 gauge needle to break up cell clumps

13. Dilute to 50 mL total volume

14. Plate 2 mL per well in 6-well plates. So one T-75 can plate into 4 X 6-well plates.

15. Allow growth in plate for 24 hours

16. Change media to chracoal stripped media

17. Allow growth in plate for 24 hours

18. If after 24 hr, cells are 80% confluent, change media again to charcoal stripped media 3 mL/well and stimulate with 30 microlitres of steroid and 5′-aza-deoxycytidine.

19. Incubate for 24 hours, harvest 4 and 5 sets for RNA analysis

a. aspirate media

b. wash cells twice carefully with cold PBS

c. trypsinize or scrape

d. add 1 mL of Trizol to each well

e. shake or place on rotor for 15 minutes

f. transfer from each well into a 1.5 mL centrifuge tube

g. proceed with RNA extraction

20. Incubate for 7 days, harvest 1-3 for protein analysis

a. transfer supernatant to 5 mL Falcon tubes

b. spin down cells

c. take supernatant and move on to protein assays and analysis

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