Several things could be contributing to a change in cell morphology and survival. The simplest thing one can do is to make sure that serum is of high quality and medium is supplemented with fresh glutamine (with time, the effective concentration of glutamine decreases in the medium). If possible, get the RPMI medium from the ATCC. If the problem still persists, purchase a new batch of LNCaP cells from ATCC and maintain as instructed by ATCC. Culture of LNCaP cells requires lot of patience, careful splitting and dispersion of cells after trypsinization.

Do you know if the black spots are moving around and growing in number within 48h? If so, these could be the culprit! It could be Archea bacteria and be controlled by using Roche BM cyclin reagents! This is another way to look at the problem!

Good luck!

I have had this problem before with my LNCaP cells - including the black dots. As Avny said, I think the black dots are probably cell debris. They can be quite an irritating cell line to work with due to their clumping and they seem to be sensitive to lot-to-lot changes in serum. This is probably something to do with the amount of serum androgens varying between batches. For me, getting a new batch of serum has resolved the problem, but the group I obtained them from suggested culturing them in 20% FBS instead of the normal 10%. Not something I have done, but it's worth a try if you've exhausted all the other options.

No, it isn't my job area.

Hi Suzy,

One way to test whether the "black dots" are a problem is to take some of your medium and add it to a sterile cell culture dish. Simply incubate it alongside your other cells in the incubator to see if anything grows. If not, it is most likely cellular debris, as others have mentioned. I have noticed this in my LNCaP cultures as well, and it does not seem to be problematic.

One concern could be that keeping them at high density can affect their growth and expression of genes. I recommend not letting them become too confluent, which can affect viability and growth rate.

Best of luck!

Hi Suzy,

For sure, LNCaP cells require a little extra care than most of other PCa cell lines. I first thaw them in T25 flask to maintain a high density and use RPMI supplemented with 10%FBS. I leave them untouched for 3-4 days and observe every day until majority are attached and then i remove the floating ones which are most likely to be the dead or damaged ones after going through the freeze and thaw cycle. Small dots are most likely to be cell debris as i also see them in my culture, however if you are still unsure you can check for the contamination as others suggested. it is also worth to test them for Mycoplasma contamination. Clumping of LNCaP cells happen between free floating, viable cells that are unable to attached to the flask. This is most likely to happen if you trypsinize the cells for longer than required. LNCaP cells don't like be exposed to trypsin for a long time otherwise they have difficulty attaching to the flask. you may also like to coat the flask with Poly-D Lysine to facilitate attachment of the cells to the flask until they recover nicely then you can move them into a normal flask. hope this helps. good luck. 

just grow it in small flast re trysinised in ebery alternative day for a week. use 15% good serum initially. Glutamine should be freash. lso supliment with pyruvate