Hi

I think you face with mycoplasma contamination.

look at:

Article Prevention and Detection of Mycoplasma Contamination in Cell Culture

https://www.researchgate.net/post/How_can_I_decontaminate_mycoplasma10

https://www.bio-rad-antibodies.com/decontaminate-cell-culture-infection-prevent-contamination-mycoplasma-removal-agent.html

http://www.protocol-online.org/biology-forums-2/posts/26501.html

good luck

Hi Chris,

Alireza might be right about mycoplasma as an additional infection in your cells; however I would like to add some points to make it much more practical:

1- Mycoplasma is an intracellular infection and you will not see that small black moving things in your culture media.

2- If you are sure that they are moving, you might consider bacterial infection for your culture. If the bacterial infection is true, the source of infection might be the operator, the culture stuffs or whatever you use in this process.

3- I would suggest to put 5ml of your media in incubator for at least 24 hours to make sure that the medium is not the source of infection.

4- For rule out of mycoplasma infection, I would suggest Dapi staining.

5- In some labs they add p/s (penicillin and streptomycin to the medium to prevent infection).

6- In terms of treatment of infection, please make sure that infection or your treatment modality do not affect your readouts.

Hope it helps.

Regards,

Zahra

Thank you for the advice! I'll test the media as you suggested.

I'm already using P/S in my media, which I know is recommended against.

Looks like I need to scrap it all and start fresh. c'est la vie...

I would do the same.

Agreed: c'est la vie

Good luck

Amended on 3 August 2018

I would like to express my opinion. Although I am full of confidence in cell culture technology, there already are many contaminating microbes.

I must sadly say that usual Immunological method is not to be utilized as a diagnosis tool. I have found that commercial antibody to detect HCV in clinical laboratory is not so precise at all; i.e., Immunoglobulin or antibody to HCV is also recognize or bind also to HAV, HBV, HEV, YMV (plant yellow mosaic virus), and CSFV/Hog cholera virus/Classical swine fever virus (our unpublished observation). Therefore, the PDMD (Protein-Direct-Microsequencing-Deciphering) method (please see file: HepG2 Fucoidan) should be used instead of notorious Immuno assay. Both your human cell-line and contaminated microbe are analyzed by PDMD method, since bacterial genetic information in Pubmed (Protein database) seems to be not perfect yet.

Hepatoma HepG2 has no bacteria due to long effect by antibiotics, but fetal hepatocyte Hc has many microbes.

Fetal hepatocyte Hc (cultured under the condition of Streptomycin (100 U/mL) and Kanamycin (100 U/mL)) already has Isovaleryl-CoA dehydrogenase, mitochondrial (Arabidopsis thaliana (thale cress)) at 25.7, 50S Ribosomal protein L4 (Caulobacter vibrioides/Caulobacter crescentus) at 16.3, Lipoic acid synthetase (Lesionella pneumophila) at 15.9, UPF0336 protein MAP_4107 (Mycobacterium avium) at 8.8, Nuclease sbcCD subunit C (Treponeme pallidum) at 7.7, Arginine N-succinyltransferase (Yersinia enterocolitica O8) at 7.3, Histidyl-tRNA synthetase (Rickettsia canadensis) at 7.1, Tetraacyldisaccharide 4'-kinase (Pseudomonas aeruginosa PA7) at 7.0, NADH dehydrogenase I subunit K (Mycobacterium tuberculosis) at 5.7, UvrABC system protein C (Mycoplasma agalactiae) at 5.7, Arginyl-tRNA synthetase (Mycobacterium tuberculosis) at 5.2, Biotin synthetase, mitochondrial (Arabidopsis thaliana (thale cress)) at 3.2, Biotin synthetase (Bacillus subtilis) at 2.7, Biotin synthetase (Helicobacter pylori J99) at 2.3 (total Biotin synthetases becomes to be 8.2), FHS 1/Formate--tetrahydrofolate ligase (Streptococcus pyogenes) at 2.2, DNA polymerase III subunit beta (Mycoplasma capricolum) at 1.6, and RNA polymerase subunit beta'/Transcriptase subunit beta' (Streptococcus mutans) at 1.1 μg/mg of cell protein, respectively. Total bacterial and plant proteins becomes to be 125.5 μg/mg of cell protein or 12.6％ among Hc cell protein.

Biotin and lipoic acid seem to be supplied from invaded bacteria and plant to animals. Therefore, symbiotic phenomenon may already be occurred by the strong link to vitamin supply and the cell growth.

Further, large virus infection may be possible.

Acanthamoeba polyphaga mimivirus (APMV) resembles bacteria on Gram staining and on size.

Hepatoma HepG2 has Uncharacterized protein L330 (Mimivirus) at 0.1 μg/mg of cell protein.

Healed hepatocyte HepG2 (cultured with Fucoidan) has increased amount of Mimivirus; i.e., Putative serine/threonine-protein kinase/receptor R181 at 1.2, Uncharacterized protein L172 at 0.62, Uncharacterized protein L237 at 0.67, Uncharacterized protein L43 at 0.16, and Uncharacterized protein L515 at 1.50 μg/mg of cell protein, respectively. Total becomes 4.2 μg/mg of cell protein.

Some human serum contains Mimivirus; i.e., serum of 1y girl (with biotin deficiency) has Putative ankyrin repeat protein R602 (Mimivirus) at 6.8, and Putative resolvase R80 (Mimivirus) at 12.3 μg/mg of serum protein, respectively. Serum of 12mo girl (with common cold) has Uncharacterized protein R641 (Mimivirus) at 3.6 μg/mg of serum protein.

Mycoplasma is too small (about 0.1 µm in diameter), and may not be possible. As assessed by moving characteristics, Caulobacter vibrioides, Lesionella pneumophila, Treponeme pallidum, Pseudomonas aeruginosa, and Helicobacter pylori are possible.

Contamination of Mimivirus may also be possible.

By the way, although HepG2 has no bacteria due to long effect by antibiotics, it seems to contain Biotin synthetase and Lipoic acid synthetase genes.

Hepatoma HepG2 (cultured without fucoidan) has Biotin synthetase (Bacillus subtilis) at 1.4, and Lipoic acid synthetase (Bacillus subtilis) at 4.7 μg/mg of cell protein, respectively.

Healed hepatocyte HepG2 (cultured with fucoidan) has Biotin synthetase (Bacillus subtilis) at 6.5, Biotin synthetase (Helicobacter pylori J99) at 0.6, Lipoic acid synthetase (Bacillus subtilis) at 3.0, and Lipoic acid synthetase (Human sequence) at 3.7 μg/mg of cell protein, respectively.

Biotin content is higher in HCC than LC in the rat liver, and this increase of biotin in HCC is due to changed Biotinidase activity via changed glyco-chains of liver biotinidase (please see file; Anticancer Res). It has also been found that gene expression of liver biotinidase does not change between HCC and LC (our unpublished observation).