Protocol for Eradication of Mycoplasma from Contaminated Mammalian Cell Cultures
Mycoplasma contamination is often reason for poor growth and viability of cell cultures. Because there is no visible signs of mycoplasma contamination, it often goes without noticing. So the first step if you notice any problem with your culture is to test your culture for mycoplasma contamination. Contact us we offer this service if you need to test your culture (ARC615).
Once mycoplasma contamination is confirmed there are few choices to take. 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. This is an expensive process and requires the use of animal. 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.
Materials:
1. MycoLytixÔ Mycoplasma Inhibitor Reagent, 1000x (ARC Catalog#113015)
2. HyMaxÔ Hybridoma Fusion and Cloning Supplement, 20x (Optional, ARC Catalog# 113004)
3. Standard cell culture media, reagents and supplies as needed
Note: Make sure the culture is in actively growing log phase while treated, cell density between 200,000 and 600,000 per mL.
Procedure:
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 10mL volume.
3. Add 0.5mL HyMaxÔ Hybridoma Fusion and Cloning Supplement, 20x (ARC Catalog# 113004). This step is optional. But HyMaxÔ provide survial factors and give better viability of culture.
4. Add 40uL ARC MycoLytixÔ 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 20uL ARC MycoLytixÔ 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 10uL ARC MycoLytixÔ 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 10uL ARC MycoLytixÔ 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 10uL ARC MycoLytixÔ 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 10uL ARC MycoLytixÔ 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.
Note: We also offer mycoplasma deconatmination as a guaranteed custom service, if required. Contact us for more information.