I'll start working with THP-1 cells but I don't know how to passage it efficiently.
Someone told me that, this cell will adhere to the surface so I have to remove the old media and incubate them with 1x DPBS for 5-10 min before passaging in order to get rid of debris. Is it true?
If the cells are looking healthy and they are more than 1.5 million/ml or if they are
clumping heavily and media is completely yellow then centrifuge out the old medium and seed them at 0.2-0.3 million/ml density with fresh medium. No need of PBS wash. If there are lot of dead cells also then give a pbs wash with low rpm centrifugation (600-800 rpm)
THP-1 will grow in suspension, unless the dish is coated with a ligand. Try and keep them between 500,000-1,500,000 cells per ml. Any less and they aren't happy. Any more and they clump and don't migrate well. To passage quickly, you can just dilute the culture in new medium, but if you have longer then, as Sameer says, spin the cells down on a low speed spin, and use all fresh medium. Try and do this at least once a week, and do check them every couple of days to see how they're looking.
What's gonna happen if I let them grow more than 1 million cells/ml of density ?
THP-1 shows tipical growth pattern composed of 4 phases:
Lag phase: cells grow slowly (after sub-culturing stress due to seeding or splitting);
Exponential phase: period of exponential growth until when the number of the cell exceeds the capacity of the medium (pH medium becomes acid);
Stationary phase: cell proliferation stops;
Decline phase: the cells number decrease and their lose viability.
Cell lines need to be maintained in exponential phase in order to assure them viability and phenotypic stability.
THP-1 growth in suspension. They needs a stimulus (LPS, ATP or PMA) to differentiate in macrophages.
I hope this help
Many thanks for your kind suggestion.
In my lab, they gently swirl the flask (T25) and then remove the old media. They said, a deteriorated cells will floating around, but the healthy one are attach to the surface so it's necessary to remove the old media before, in order to get rid of debris and deteriorated cells. After removing the old media, they add the new media into the flask and pipetting in order to detach the cells from surface then pelleting them and reseeding.
Now I'm a bit confused.
you are wellcome!!
In order to replace the medium you have to harvest the cells from (T25) and put them into tube (15 mL volume), centrifuge 800 rpm, wash twice with PBS (centrifuge 800 rpm), resuspend them in new medium or filter, count (check the viability) and seed them.
THP-1 attach (differentiate) only in presence of stimuli such as LPS, ATP or PMA.
Hi, I have read this discussion and I have a question regarding the evaluation of TNFalpha production after THP-1 stimulation with LPS.
For what i have understood, after exposure to LPS cells differenciate to macrophages and the adhere. In order to detect the Cytokine, should I just centrifuge and take the supernantant?
could I do the same also for the ctrl cells that have not received LPS (in this case all cells in suspension)?
Another question, how do you perform viability test on THP-1? did you use the MTT test?
thanks for all your help!
- Badges
- Science method