Protocols

Protocol for eradication of mycoplasma from contaminated mammalian cell cultures

Mycoplasma contamination is often the reason for poor growth and viability of cell cultures. Because there is no visible sign of mycoplasma contamination, it often goes without notice. So the first step if you notice any problem with your culture is to test it for mycoplasma contamination. Contact us — we offer this service if you need to test your culture (ARC615).

Once mycoplasma contamination is confirmed there are a few choices to make. The best choice is to discard the culture, if you have a backup of certified mycoplasma-free culture. If you have a heavily contaminated culture and would like to rescue it, one option is to grow the culture in mouse ascites — the mouse immune system will eliminate mycoplasma, though this is an expensive process and requires the use of animals. The third choice is to treat your culture with anti-mycoplasmotics for several generations until you have a clean culture. This is the easiest and cheapest option. Here is the protocol for doing this.

1. Determine viable cell density of the contaminated culture.
2. Centrifuge cells, and plate contaminated cells in a new T25 flask at 200,000 cells/mL in 10 mL volume.
3. Add 0.5 mL HyMax™ Hybridoma Fusion and Cloning Supplement, 20x (ARC Catalog #113004). This step is optional, but HyMax™ provides survival factors and gives better viability of the culture.
4. Add 40 µL ARC MycoLytix™ Mycoplasma Inhibitor Reagent, 1000x (ARC Catalog #113015) to the culture. This will give a 4x concentration.
5. Incubate for 48 hours.
6. Determine viable cell density of the contaminated culture. Replate at 200,000 cells/mL as before.
7. Add 20 µL ARC MycoLytix™ Mycoplasma Inhibitor Reagent, 1000x (ARC Catalog #113015) to the culture. This will give a 2x concentration.
8. Incubate for 48 hours.
9. Determine viable cell density of the contaminated culture. Replate at 200,000 cells/mL as before.
10. Add 10 µL ARC MycoLytix™ Mycoplasma Inhibitor Reagent, 1000x (ARC Catalog #113015) to the culture. This will give a 1x concentration.
11. Incubate for 48 hours.
12. Determine viable cell density of the contaminated culture. Replate at 200,000 cells/mL as before.
13. Add 10 µL ARC MycoLytix™ Mycoplasma Inhibitor Reagent, 1000x (ARC Catalog #113015) to the culture. This will give a 1x concentration.
14. Incubate for 48 hours.
15. Determine viable cell density of the contaminated culture. Replate at 200,000 cells/mL as before.
16. Add 10 µL ARC MycoLytix™ Mycoplasma Inhibitor Reagent, 1000x (ARC Catalog #113015) to the culture. This will give a 1x concentration.
17. Incubate for 48 hours.
18. Determine viable cell density of the contaminated culture. Replate at 200,000 cells/mL as before.
19. Add 10 µL ARC MycoLytix™ Mycoplasma Inhibitor Reagent, 1000x (ARC Catalog #113015) to the culture. This will give a 1x concentration.
20. Incubate for 48 hours.
21. Take a sample of culture and test for mycoplasma by ELISA or PCR.