The black dots could indeed be due to bacterial contamination, as indicated by Vaibhav. Good general advice about cell culture.

If the cultures are not contaminated with bacteria, another reason to look for is mycoplasma since they can alter the cell morphology, which may look "punctuated". This happens very easily any time cells are in the same incubator as mycoplasma-contaminated cells. In our lab we treat ALL cells with a mycoplasma removing agent as soon as we receive them, no matter where the cells come from. It takes a week and is very effective. Mycoplasma contamination would also explain why you need to feed the cells so often. You can either test the cells for mycoplasma contamination, or just treat them for a week. However if you do have mycoplasma, all your cultures need to be treated, and the incubator and hood thoroughly cleaned. 

LNCaP cells: we feed them twice/week in the medium that is recommended by ATCC. We use phenol red-free medium (all androgen-dependent cells should be grown in phenol red-free). Complete medium is Phenol red-free RPMI, high glucose (normal RPMI is at 2g/L glucose) = add 1.25 g glucose for 500mL medium (2.7 ml of 45% glucose solution),  glutamine and peni/strepto,  10% FBS, 10-8M DHT.

Note that contrary to what is commonly believed, normal FBS does not contain enough androgens, and you need to complete with DHT 10-8M to keep the cells healthy and androgen-dependent. On the other hand, phenol red does interefere with AR, which is why we use red-free medium.

If you passage the cells before they become clumpy (typically 60-70% confluency as indicated by Martin), you will not have too many problems. But these cells just do that. Once they become clumpy, there is not much you can do (I aspirate and discard the clumps by hand with a glass pipet before adding trypsin). We usually passage these cells at relatively low density only once a week because of their slow growth (doubling time is about 40hrs), with one medium change in the middle of the week.

Hope this helps. Good luck!

I faced the same problems when I was working with LNcAP cells. LNcAP Cells are very sensitive for physical stress. The black dots you see are actually cell debris/dead cells. For a 100mm dish 1.5-2 mL of tripsin-edta would do fine. Do not tap the dish, that will be physical stress for the cell, and they will also form clumps.  gently mix the cell suspension with pipetting. After plating the cells in fresh plate/dish do not disturb the plate for at least 24 hours.

Hi, I think these dots are not a worry. Adherent cells releases microparticles, this is the case you see if you seed more cells. Solution to this I think is whenever you split your cells give 3-4 washes of PBS and seed around 1.5-2 million cells homogeneously with fresh warmed media. After seeding just shake the plate 2-3 times slowly, you will see even distribution of cells. Generally pipetting up and down is not recommended because it might break your cells and as a result you see adhered cells in a group. Seed less cells and shake the plate, you will not see any clumps. For 10 cm plate, I use 2.5 ml of trypsin that covers the entire plate in one layer and same amount of media I use to inactivate trypsin. Time actually depends from cell line to cell line for incubation with trypsin. If your cells are more robust you can keep it at 37 degree for 5-7 minutes and then you can do the gentle tapping because incubation will loosen the cells and gentle tapping will make them non adherent. You don't have to change the media every 24 hrs, that is required when your cells are very fast growing and you see lot of cell death (change in media color). 

You main also try to add some FBS (around 5%) while collecting the cells after trypsinization as it will lower cell-cell interactions and should consequently reduce cell clumping.

never faced the problem with black dots in LNCaP cell culture.

To distribute equally the cells throughout the plate, i usually "draw" 8 with the plate. This motion usually makes the cells to be distributed equally. 

1ml of trypsin is usually enough to cover the cells in 10cm plate.

to prevent clump formation, you can use 1ml pipete, after spin

I will check for mycoplasma first if you see black dots. LNCaP grow best at high seeding density, I will say 5x10^5 per 100-mm plate at the least. Use 1 ml trypsin for 100-mm plate for 1-2 mins, neutralize with 1 ml of serum-containing medium and pipette 10 times with a P1000. Can also use a 40-70micro filter to remove clumps if single cells are necessary. They grow as "spheres" at high cell density and never reach confluence.

I agree with Andrew M Chan....check for Mycoplasma contamination...you can add 1-1.5 ml trypsin and tilt your plate and remove trypsin quickly there after. Then you can incubate your cells for 1-2 mins in 1 ml trypsin. Try to monitor the cells in 30 second interval and stop trypsinization by adding serum containing media. Repeatedly pipette to remove cell clumps and sub-culture or count them as per your experimental needs. This way your cells will remain viable and will trypsinize efficiently..

All the best.

Thank u all for replying and for your valuable suggestions. I will follow these and let you all know whether the problems go or not.  Mr. chan and sachin, mycoplasma is so small that it cannot be seen by light microscopy. So how can you say that ,these dots can be mycoplasma

I am not saying that these dots can be mycoplasma,,,i am just advising you to check for the contamination in case the growth behavior of your cells is not appropriate.

Well due to the slow growing nature of LNCaP its little bit more tedious comparing to other Prostate Cancer Cell lines.Very first thing while maintaining and culturing LNCaP is that what ever lab apparatus like pipette tips etc u use shuld be autoclaved properly and the culture flasks used should be sterilized and new.From your problem I culd make out  that there are handling issues which are very basic but extremely important to long term study while using LNCaP. Initially I also had the same issue while working with LNCaP , the black dots are totally the result of contamination while handling or might be due to changes in temperature(37.1 C) during incubation.Normally the LNCaP cells are very clear to see under microscope apart excessive trypsinization also results in unresponsiveness of the cells.My suggestions for your  problem is that maintaining sterilization and aseptic environment  will solve your all your problems you mentioned.Here are few points that you should take care of.

1-Keep all your Lab Apparatus in Laminar flow 15 mins before while turning on UV radiation, which will minimize the contamination rate due to Lab App.

2- Culture Media,Trypsin etc should be sterilized and must be opened only in Laminar flow.

3.Same way your culture flask,autoclaved pipette tips and single use pipettes  should be opened only in Laminar flow.

4-Also check your Laminar flow is maintaining the air flow properly.

5-Kindly do not use culture flasks and usable pipettes more than once. 

6-Discard flask with cell if u c cloudiness excessive clumps that is the surity of microbial contamination.

.Try following these steps carefully n it will solve all ur problems  if not add Penicillin and streptomycin solution in your culture media before you culture any new cells.

following you will achieve almost uniform distribution of cells on your culture plate as well.Let me know if you further have any problem with it. My Email ID:               [email protected]

I am also attaching one ov my publication which include some related aspects

Many Thanks n All the best 

Article Curcumin Restores Glutathione-S-Transferase Activity for LNC...

I agree with A Chan, too. Use 1 ml trypsin for 100-mm plate for 1-2 mins, neutralize with serum-containing medium and pipette 10 times with a P1000. Sometimes, I do not use trypsin. Remove medium, add 1 ml fresh media with serum and gently tap until that cells are in suspension. Then, pipette 10-15 times the cell suspension gently with a P1000. Seeding to low density. 

These cells grow in 8% FBS and 1% antibiotics (100 units of penicillin and 100 µg streptomycin) at 37o C in a 5% CO2 atmosphere. Medium replacement is performed every other day and cell passage is done by trypsinization when cell confluence reached 70%. Then you can seeded at 1.5 x 103 cells/cm2; for experimental conditions, the seed stock is replaced after 12 passages. Is possible to have individual cells but eventually grow in clumps, so do not worry it's part of their behavior. 

Black dots can either be fungus, clumpy cells, or air bubbles. It's very likely the 2nd or 3rd issue. Best practices for tissue culture is kinda weird, everyone says to passage at 80-90% but realistically you can and should passage earlier than this.Typically between 60-70%. If you wait too long, your cells will basically freak out. So be on the safe side and just passage early. If you do this consistently, your cells will continuously develop nice monolayers.

The black dots could indeed be due to bacterial contamination, as indicated by Vaibhav. Good general advice about cell culture.

If the cultures are not contaminated with bacteria, another reason to look for is mycoplasma since they can alter the cell morphology, which may look "punctuated". This happens very easily any time cells are in the same incubator as mycoplasma-contaminated cells. In our lab we treat ALL cells with a mycoplasma removing agent as soon as we receive them, no matter where the cells come from. It takes a week and is very effective. Mycoplasma contamination would also explain why you need to feed the cells so often. You can either test the cells for mycoplasma contamination, or just treat them for a week. However if you do have mycoplasma, all your cultures need to be treated, and the incubator and hood thoroughly cleaned. 

LNCaP cells: we feed them twice/week in the medium that is recommended by ATCC. We use phenol red-free medium (all androgen-dependent cells should be grown in phenol red-free). Complete medium is Phenol red-free RPMI, high glucose (normal RPMI is at 2g/L glucose) = add 1.25 g glucose for 500mL medium (2.7 ml of 45% glucose solution),  glutamine and peni/strepto,  10% FBS, 10-8M DHT.

Note that contrary to what is commonly believed, normal FBS does not contain enough androgens, and you need to complete with DHT 10-8M to keep the cells healthy and androgen-dependent. On the other hand, phenol red does interefere with AR, which is why we use red-free medium.

If you passage the cells before they become clumpy (typically 60-70% confluency as indicated by Martin), you will not have too many problems. But these cells just do that. Once they become clumpy, there is not much you can do (I aspirate and discard the clumps by hand with a glass pipet before adding trypsin). We usually passage these cells at relatively low density only once a week because of their slow growth (doubling time is about 40hrs), with one medium change in the middle of the week.

Hope this helps. Good luck!

Removing the clamps: easy way when you gently centrifuge after trypsinazation, remove the superntant, then flak with finger for few times to dissociates the precipitate of cell. hope it will work for you.

I want to know that for the attachment of the LNCaP cells, whether i have use gelatin or not 

Has anyone tried using BGS serum (Hyclone) instead of FBS for growing LNCaP cells? 

Hi Monica.

Yes I did try n its equivalent replacement to FBS. However no major differences were observed in LNCaPs growth but it culd add up a little uniqueness to your work.

Thanks Vaibhav, I will try using BGS as I am starting to work with LNCaPs.

We use RPMI medium supplemented with 8% FBS and penicillin-streptomycin

I would test for mycoplasma first. Apart from that, LNCaP needs to be seeded at high density (~30-40%), especially when you first thaw them. Split cells before they clump since they don't go to confluence. Use low trypsin (0.05%) VERY briefly (15 sec) since LNCap cells are highly sensitive to trypsin so neutralizing with full serum containing medium immediately and pipette up-and-down to break up cells.