Hello Anshuli,

That's an interesting question. I have performed MTT assay or Crystal Violet Assay but have never considered the effect of Pen/strp on IC50. I dont think if there is any study about this. However, there is study that shows that PenStrep treatment can significantly alter gene expression and regulation in a common liver cell type such as HepG2, advocating that antibiotic treatment should be taken into account when carrying out genetic, genomic or other biological assays in cultured cells.

https://www.nature.com/articles/s41598-017-07757-w

I will however be following this question.

Muhammad Zawwad Raza

Hi Muhammed Zawwad Raza,

Thanks for replying and sending the article! I did MTS assay using pen/strep only once on two cell lines and it gave me interesting results, for one cell line the IC50 was within the expected range but for the other it was significantly low. I am not sure if it was because of the antibiotics or other factors, but I haven't used them again for MTS. But I did wonder if antibiotics were the culprits and if some cell lines are more susceptible to them than others.

It is also interesting that you mentioned HepG2, as this is the cell lines I am using besides other liver cancer cell lines.

Anshuli

Hello Anshuli

I think using of 5% antibiotics have no effect on the cell line. However, you can do MTT assay on your cell lines that incubated with antibiotics as a control.

wishes

Ahmed

Hi Ahmed,

I use 1% antibiotics for the routine culture. I could do the MTS with or without antibiotics but it requires some extra efforts as I'll have to do it for all the cell lines I'm working on (and may be repeat 2-3 times), so I wanted to know if anyone has done anything similar and also, if people use antibiotics when doing MTS/MTT assay?

Thanks,

Anshuli

I do not use antibiotics for HUVEC cells but I still got variable results of MTS for treated cells. I normally let cells became 80% confluent, then treated them for 48 hrs prior to MTS. I repated three times and twice cell viability slightly decreased (about 10%), once increased 60-70%. I am using Promega MTS assay. I cannot explain these results but I will repeat it.

PenStrep as well as many other antibiotics affect ETC and therefore OCR of mitochondria (hence cells) as mitochondria resemble prokaryotic cells for which antibiotics were originally designed by nature. As such, even PenStrep may affect MTS readout.

Although at low concentrations PenStrep does not change OCR, this can be misleading because in reality it does. The reason you do not see much of a change is that the cells adopt to antibiotics by metabolic reprogramming. I always advise my students to perform a simple exp - take freshly isolated skin fibroblasts and measure OCR in the presence or absence of PenStrep (same dilution as for the routine cell culturing) - you will see difference.

If you want to see such difference in the cell lines already grown in the presence of PenStrep, remove antibiotics and culture them for 2-3 weeks - this time is sufficiant for the mitochondria to undergo turnover - then you will start seeing the influence of antibiotics again.

Finally, may I provide you with few more papers:

doi: 10.1016/j.canlet.2016.09.023. you may see OCR change in the presence of some antibiotics;

doi: 10.1002/med.21459. you may see a pic showing why antibiotics affect mitochondria but not the rest of the eukaryoptic cells;

doi: 10.1074/mcp.RA118.001102. here you may see how certain antibiotics create mitochondrial dysfunction and suppress cells that are prone for OxPhos.