Hi,

the number of passages should be initially provided by the seller. The senescence process is related to the number of passages in culture and the only possibility is to renew the cell line (by thawing a new aliquot). Usually 30 passages are the max and then it is better to trash the cells.

Regards.

Initially you should keep and eye on them, I would count the cell number everyday. They don't like to be split to thin. They are good for at least a month but once you realize that they are growing faster or slower than usual it is best to to thaw a new vial.

Hi Esmeralda ,

THP-1 cell line is the cell depending on type of use you do. The Above said comment by maurizio is correct but it can be used upto 25th to 27th Passage , If you using them for Biomarker Validation And up to 20th Passage if using them for Differentiation and other relative uses. Be Careful in doing passaging as if it starts giving false positive/Abrupt results in Assays/Experiments.Please discontinue the current culture and Revive the fresh batch. Please let me know if you need further information.

Regards

Manvendra.

We used THP-1 in RPMI with 10%FCS. Only in the very beginning the THP1 cells had problems to grow well. After some passages they grew very well. You can keep the cells in culture for a long time. Depending on your design we usually take a new aliquot after 25 passages.

I culture the THP-1 cells in 10ml RPMI1640 + 10% FBS P/S media. 1:10 split every 5-7 days. maintaince the culture for no more than 30 passages and thaw a new vial.

THP-1 cells usually grow in suspension unless you stimulate them with a mitogen such PMA, at that point they adhere and differentiate. These cells does not go under senescence if they are not stimulated but just divide and you have to split them at least twice a week. after differentiation 10 passages are enough the you have to change to a new non differentiated cells.

Thank you all,

I appreciate your answers. They're quite helpful I'll be watching my cells (I just got them from ATCC). I believe this is a silly question, but as I already said I'm new at this and I wonder if every split is to be considered a passage or it is related to freezing-thawing batches. Once they're differentiated they're not suppose to proliferate right?

Thanks again

Esmeralda.

Passage number initially mentioned in the data sheet. The senescence process is related to the split ratio and time as well as growth condition. For better efficacy you can trust up to 12 passage and characterize the cell line. Usually it will safe up to 28 passage.

Yes Esmeralda, every split is considered as a passage. Do not dilute your cells too much. A rate of 2.5 x 10E5 cells/mL is OK and you have to split the culture twice a week. Thaw a new sample when the cell morphology change or when your results are differing from the results in your previous experiments.

hello, 

We only use THP1 cells uptill 5 passages. 

I am culturing THP-1 cells in suspension from last 15 days but at passage 5 suddenly they started to adhere on T-25 flasks without any stimulation (PMA or MCSF).

I am really clueless about this.

can anyone tell me what might be the reason of this...

ok thank u so much Cristina... 

so what you people did to solve this problem..

thank u cristina...

we work with THP-1 and U937 cells. When THP-1 cell start to attach means that they get activated by something to differentiate in more macrophage like cells. There might be something in your medium or that the culture dishes or flasks are different. I would try fresh medium and maybe different culture material. The serum you are using for your culture medium is critical also.

I can suggest you three things that you may consider to check.

-One is the FBS, make sure you are using one from a trusted vendor, I am using Gibco and heat inactivated  one

- mycoplasma could also be a big issue.

-for the media I used to use RPMI  media.

Good luck