I am experiencing some strange problem in some adherent cancerous cell lines. I have observed that these dots only adhered to cells for first 2 days after passaging, but these dots then start to become visible in the media also. The number of dots increases after 3-4 days if cells are not sub-cultured.
The cell lines grow properly, there is no change in pH of media or turbidity. I changed FBS, Trypsin, Media etc. i also thawed frozen vials but the problem still persists.
After washing 4-5 times at 500 RPM the number of dots decreases significantly but after culturing the dots start to appear again. We did DAPI for mycoplasma but it came Negative. Treated with PLASMOCIN for 1 week.
It is clear for me that the dots observed by Mohammad are not mycoplasma because although they can be seen through the microscope, they are not stained by DAPI as Mohammad stated. The release of microparticles (which are different from debris) by adhering cells is a well described and interesting phenomenon, Mohammad, don’t worry about this.
This sounds like contamination of some sort. The most likely cause if you have the common antibiotics in your media is mycoplasma. This is a common contaminant in cell culture. I suggest that you do PCR or other mycoplasma test of your cell lines and then treat with cipro or other commericial mycoplasma treatments.
If the size of dots is different they might be cell debris from high cell death.
Check out this literature for your problem:
http://www.ncbi.nlm.nih.gov/pubmed/19926304
It might help..
Hi Mohommad, split the cell thin and then let them grow to the point where you begin to see the "dots" and then let the plate sit for 5 days without changing the media. If the pH changes, as indicated by a color change in your media, then you have contamination. Be sure to set up a control plate that has cells without this "dot"-like contamination (at the same cell number of course). Best of luck.
I agree with George and Syed; it could either be mycoplasma contamination or cell debris (less likely though given that your cells, morphologically and growth-wise, seems healthy). I would definitely perform a mycoplasma test before carrying out any experiments. I would also put the culture flasks in a separate incubator, just in case (your culture room might have adesignated 'mycoplasma-risk' incubator). All the best.
Check this out... may be this relates to your problem
http://www.ncbi.nlm.nih.gov/pubmed/19926304
Got black swimming dots in your cell culture? Identification of Achromobacter as a novel cell culture contaminant.
Gray JS, Birmingham JM, Fenton JI.
Abstract
Cell culture model systems are utilized for their ease of use, relative inexpensiveness, and potentially limitless sample size. Reliable results cannot be obtained, however, when cultures contain contamination. This report discusses the observation and identification of mobile black specks observed in multiple cell lines. Cultures of the contamination were grown, and DNA was purified from isolated colonies. The 16S rDNA gene was PCR amplified using primers that will amplify the gene from many genera, and then sequenced. Sequencing results matched the members of the genus Achromobacter, bacteria common in the environment. Achromobacter species have been shown to be resistant to multiple antibiotics. Attempts to decontaminate the eukaryotic cell culture used multiple antibiotics at different concentrations. The contaminating Achromobacter was eventually eliminated, without permanently harming the eukaryotic cells, using a combination of the antibiotics ciprofloxacin and piperacillin.
Hi Mohammad,
What type of cells are you growing? We use a breast cancer cell line that produces a lot of floating bodies/specks that are not a contaminant. The culture is also healthy and negative for mycoplasma. So maybe it's the type of cancer cell line that you are using and may be something the cells themselves are producing.
Hi Mohammad,
Everyone is correct. Could be cell debris, could be mycoplasma, could be another contamination. Is the media turbid? The dots are swimming? This could be a bacteria.
The mycoplasma is the most common contaminant in culture and the best way to catch it is when you use vacuum pump to aspirate the media since in the tubing a reflux is generated and the mycoplasma could swim into your flask if someone else with contaminated cells aspirates his/her culture before you and didn't clean with bleach the tubing (it is not enough to change the aspirating pipet!!).
The best way to keep your cells clean from the beginning is to discard media in another recipient using a sterile regular culture pipet.
The mycoplasma can affect your culture depending on what you are doing. I remember a group in France that have a contaminated culture on purpose because the protein that they were studying was overexpressed in contaminated cells.
Also, if you try to measure (3H) thymidime incorporation, the mycoplasma will degrade the thymidine to thymine (3H) resulting in reduced incorporation to your proliferating cells.Mycoplasma was my nightmare 20 years ago since we thought that we discovered a nucleosidase in our cells similar to the one platelets express.
Once you have it is very difficult to eliminate, although some companies claim special reagents to do it. In my experience you can clean it but it can come back. If your cells are unique and very valuable and you cannot start over, clean it with any of the products and check it periodically for the contamination to re -appear. Good luck!
I agree with Seyed Mahmoud Hashemi. I had the same problem when suddenly dropped the level of CO2. I was doing research on mycoplasma. It was OK. After connecting another bottle of CO2 everything returned to normal
Hello,
For mycoplasma, if you have antibotics in the medium, it's better to repeat the test with cells cultured in the absence of antiobiotics.
The cells may be in stress. So you can test all the medium components one by one.
If this problem was observed after thawing, test if it's related to your cell bank.
Good luck
Hi Mohammad,
It could be cell microparticles which are released via membrane blebbing even by healthy cells. This microparticles could be responsible of cell to cell exchange and are currently observed in adhering cancer cell culture.
Hi Mohammad, i got exactly the same problem with my MDA-MB-231 and MCF-7 breast cancer cell lines. Those small black dots did change the behaviors of cells and their response to treatment with telomerase inhibitors. So i tried to treat the cells with different antibiotics but i did not succed, so i discarted all the cells and ordered another ones from ATCC.
Good luck
Hi Mohommad, As i got same Black dots with my MCF 7 CB5 and CMV.Split the cells and wash twice with PBS sterile then incresae the antibiotic concentration upto four folds which is not lethal to cell growth ..i calibrated the penstrep antibiotic concentration,you can use high concentration,to get rid off contamination.
Could be particulates from the serum (flocculent). Do you filter your serum? Do the cells grow very rapidly - cell debris?
Also look at the possibility of cryoprecipitates from the serum. Thawed fetal bovine serum does have some cryoprecipitates in it.
From the description of you problem, I rather think that your cells are producing the debris. Some cells do that. I have stomach cancer cells that seem to loose their granules during growth.
If you want to confirm fast and unexpensive the possibility of infection, do a DAPI staining of fixed cells and look in a fluorescent microscope. If it is an infection, you will see a lot of "blue" dots corresponding to your "dots" swimming in the media.
DAPI stains the DNA, so BE CAREFUL. Do not stain too long or you will stain the mitochondrial DNA and your will get a false positive. The mitochondia DNA will look similar to a mycoplasm contamination.
Good luck!
Hi, I consider it as contamination and I suggest you keep your cells for long time, may be 5-6 days and if they die, you need to change the source of cells. There are lots of interpretation on them from mycoplasma to........, where I personally agree with the mycoplasma, but once you have them in your cells and they keep on growing, I suggest to go for a new sample.
I have seen this before with primary cells as well. its likely cell debris. however, I also add the fungizone to culture media as a precaution against yeasts.
Mycoplasm contamination is a common problem for cell culture, and it can not be detected with only DAPI staining. You have to use PCR based assays to be more precise. However, these dot like structures are also seen in some cell types only and do not seen others, which mplies that these are cell type specific, therefore can not be contamination. If so, these structures are made up of cells themselves. Follow your cells' growth kinetics, morphology very carefully before discarding. Use precisely heat inactivation for your serum source by yourself (do not use preheat inactivated serum), growth media with right glucose conc., change brand of your plastic ware and go for flasks, if possible. On the other hand, if your lab is class A and has microbiology laboratories on the same floor, there is no way out to get rid of stress activating factors for your cancer cell lines. This is our observation and we are currently going to class B lab facilities. Good luck.
Hi I would test for Mycoplasma Infection and treat cells accordingly. I have experienced similar findings and when I have tested for Mycoplasma they have been highly infected. It reduces after splitting your cells and then increases as the cells grow and divide. I routinely check for mycoplasma with all my cells. Check for mycoplasma when cells have been left growing for at least 2 days after splitting. Good Luck!
If there is no change to the way the cells behave (can you be sure of this? Have you got cells that don't have the dots to compare?) then it is unlikely to be mycoplasma.
However, like mentioned above, if you plate some cells, fix after a couple of days and stain for DAPI you should be able to tell. The mycoplasma will be positive for DAPI..! There are fluorescence based and PCR based assay commercially available too..
Personally it sounds like it is just cell debris though. All my cancer cell lines produce debris which increases over time and does not affect their growth. If you do some standard live cell phase contrast microscopy, you can actually watch them shedding this debris all the time! It's quite interesting to see how much they lose...!
PS It's always a good idea to routinely check for mycoplasma anyway. This should be a standard procedure in all labs!
a few things seem to be common:cell debris, precipitates from the media, very early fungal infection. Overall, it seems harmless, but you could always do a control with media alone in your incubator for a few weeks to be sure.
could you send a picture of your black dots to me?
A note to Kermal-- myco CAN indeed be detected "only" with the DAPI method, which has the advantage of detecting all strains, whereas PCR-based ones only detect specific "most common" strains. The DAPI method is limited only by the quality of your microscope and your technique. Properly done, one takes media from the suspect infected lines and use it to infect a negative standard cell line. That should work for most types of suspect "infection" (unless it's a virus with specific cell tropism).
In this case, assuming that myco was ruled out, besides "debris" another explanation is exocytic vesicles- we work with a neuronal line (NSC34) that produces these abundantly. One could take the "black dot" media and add it to cells that don't produce "black dots" to see if it is infectious or just normal. Good luck!
To Nancy:
Dear Nancy, I would be careful with doing the typical "Take Black dot media-add it to new cells- see if they reproduce" method, since there are cells that could detect this foreign debris as abnormal and start a whole casade that causes the production of more granules because the cells are totally stressed. It could give a false positive.
And a little correction of what I proposed: DAPI will tell you if there is "other" DNA floating around in compacted forms, but off course, if you want to be sure if it is mycoplasm, you have to do PCR. The DAPI just saves you time, since I will not keep any cell having a contamination (if I have more sources for the cells, of course!). The process of cleaning them up could be successful, but you cannot be 100% sure that the cells did not change during the process. After all cancer cells are well known to adapt to changes by changing themselves.
Have fun!
It is clear for me that the dots observed by Mohammad are not mycoplasma because although they can be seen through the microscope, they are not stained by DAPI as Mohammad stated. The release of microparticles (which are different from debris) by adhering cells is a well described and interesting phenomenon, Mohammad, don’t worry about this.
I have experienced the same problem. It is very estrange as these dots do not swim but seem to vibrate. Maybe this article is useful for you: http://www.sciencedirect.com/science/article/pii/S1045105609001304
This is something we observe some times with primary cells, especially cancer ones, with high population doublings.
If the dots differ in size it is more likely to be cell debris. If you check your cells under a good phase contrast microscope i.e. a time lapse, you can actually watch them producing debris , there is an online video of HeLa cells divining and at the same time you can see the cell vacuoles and cell debris produced as well.
However, like mentioned by other colleagues, it is advisable to always check for mycoplasma. You can plate some cells, fix them after 2-3 of days and stain for DAPI you should be able to tell if there is other DNA (mycoplasma) floating in there which will fluoresce, if not then it is just cell debris.
good luck
I doubt this dots are mycoplasma. You can net see mycoplasma under a microscope without DAPI, they are too small. One sign of cell contaminating with mycoplasma could be cells producing lot of debris. Therefore I would test for mycoplasma contamination after at least 5 days without antibiotics and without medium change nor trypsinisation.
In between, one think you should look at is how these debris move. If they have brownian mvt, then I would suggest it is not alived. However, if you see them moving in clear straight direction, then could be lived organisms.
One test i would suggest, is to put next together some wells with cells and medium and other with medium only. If you see these dots only in the wells contining cells, then i would say they are debris. If you see them in medium only containing wells, then It is something containing in your medium (probably serum) that aggregate and precipitate when at 37 C. In both cases, i would not worry.
hope it helps
There are few possibilities.
1. There could be a mycoplasma contamination in your culture.
2. These could be cell debris from excessive growth (if cells grow very fast)
3. Because of antibiotics.
I also experienced this problem once. and that is the first thing I would suggest to check for Mycoplasma contamination and do culture without antibiotics or use alternate antibiotic.
It seems that what you are observing are exosomes. We, actually, had a very similar problem with our glioblastoma cell line and spent initially quite some time to exclude infection. Then I found many papers describing the phenomenon. Certain cell lines, especially glioblastoma and many primary cells produce exosomes containing mitochondrial proteins and DNA and other cellular material. There is quite some research going on the subject and it seems that exosomes are important means of communication between cells.
Just check PubMed and do not worry, because your nasty dots can turn out to be a nice new project!
Check the FBS or FCS you are using. If you had heat inactivated it, did you do it at 58 degree for 30 mins or more? As precipitation can occur in serum may not alter the properties of cell growth.
I too had similar experience with my cells earlier .They are fine as long as culture is free of mycoplasma. They are cell debris ..
hello MD Ishaq,
As per my experience it is very difficult to come to any conclusion even by seeing flask in higher magnification microscope also.To rectify these issue, you can check out these points:
1) It can happen either because of Serum you are using or because of cell debris.As u did DAPI and found it negative then there is less chance of micoplasma contamination.
2) Another reason would be over trypsinization, just be careful and once you try with less inocubation time what you are using while trypsinization now.
3) If it is cell debris then In between two passaging,u can go for fresh media change followed by PBS washing as well.
Best luck!!
Thank you all for your valuable comments. My cells are ok now although small no of DOTS are still there. I am working on it and when i will completely get rid of those dots i will write the whole procedure. Once again thank you.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2849847/?tool=pubmed
This is a paper that characterizes the black particle phenomenon.
How long do you need to observe the dots? I observed these black tiny dots several times but only when I left my medium unchanged for more than 4 days, just before I needed them for downstream use. Thinking back, I could have already contaminated my cells from the beginning but I changed medium every 3-4 days so it was unseen. We are not sure what kind of contamination that is as we never test for contamination ans I was the only one in my lab facing this recurrent contamination.
Oh, but the cell lines were given from another lab.
Hi, me too facing the same problem. But in my case the black particle are moving. I want to here from you, complete process, how u get rid of these dot particles. Check the following link once, it may helpfull for u.
Http://www.protocol-online.org/forums/topic/21330-black-dots-contaminant-phenonmenon-helpful-paper/
all the best
Hi,
I also faced similar problems with m culture. May I know which cell lines you were culturing? I have read that small movement of the dots can occur due to Brownian movement. But in that case how to distinguish wether the moving small dots are just due to Brownian movement or is it really contamination.
try to stain with DAPI staining, if it got stained with DAPI, it means that it would be mytoplasma. Although you did try stain with DAPI, but I'm still positive it's mytoplasma. try to treat it as mytoplasma, it will go away.
Guys check co2 levels of your incubator. I had the same issue and noticed it was co2 level in my incubator was very high, although the reading was 5%. It's just because the sensor was broke and actual Co2 level was very high. Changed incubator and no black dots now.. Hope this will work for u.
1 minute ago
Hi Ishaq..........
these are indeed bacterial contamination.........
Please start a fresh by autoclaving each & everything. If not done they will grew in numbers & destroys your cells.
I have suffered for 6 months with this contamination.
for future You can use GENTAMICIN as suggested by the article
[http://www.ncbi.nlm.nih.gov/pubmed/19926304 ]
'''''''Got black swimming dots in your cell culture? Identification of Achromobacter as a novel cell culture contaminant ☆
Jennifer Sue Graya, 1, Janette Marie Birminghama, 1, Jenifer Imig Fentona, b, "''''
There are a lot of factors few which I have information may be help for you. Dark dots in cell culture may be the cell debris, if it, then the darken particle will be of differing in size. But if these dots are of same size and there is also amplification observed this indicates there must be some bacterial contamination. There is also one more another case for it, that is granulation in cell culture due to stress in your culture which also appears as dark dots.
Just add few drops of culture medium onto Nutrient agar plate and incubate at 37 C to rule out any contamination.
Even if you don't see a change in media color it can still be contamination. You should test this by transferring a few microliters of "contaminated" media to a new flask containing either sterile media alone or sterile media + “clean” cells. On the other end, it could just be cell debris.
Metabolic processes continue in cells during cell culture and I believe that the dark dots you are referring to are products of these processes or remnants of cells. To rule out the suspicion on contamination, try to collect several aliquots of the sample and plate them out in nutrient agar.
Hey Mohamed,
I have seen those dots before and initially I got worried - they normally originate from the serum used in the culture media. Such sera may have stayed for long since collection or were heat-inactivated longer than 1 hr or at higher than 56 degrees Celsius temperature. If you continue long enough with your cultures, you will notice, when you change to a new serum batch that is less "fatty", that the dots will disappear with time.
I do not think they are a bacterial contamination as someone suggested earlier - bacterial contamination in cultures are very hard to hide for more than 24hrs in the nutrient-rich culture media people use. Mycoplasma can hide for a while but with time, you will notice changes in cell growth or cell phenotypic deviations or even in its chromosomal characteristics.
Good luck.
The black dots you see are probably signs of dying cells. We had such a problem where the cells looked healthy but with these dark dots inside the cells. It looks as if these dots appear when the cells are facing stress in some form. The probable reasons are low density of cell seeding, low serum levels, buffering of the media etc. I have also seen these black dots before the start of a fungal or mycoplasma infection. There was no change in the pH of the media. However, the persistence of these dots leads to cell death without any visible contamination if the media is left unchanged for about 10-15 days. I had started a separate culture using a fresh seeding with 20% FCS reducing it to 10% gradually and I could get rid of these dots. I maintained the culture with 10% inactivated millipore filtered (important) serum afterwards. So my suggestion to you would be to check each parameter separately and identify the stress factor.
Good luck.
No worries. Its definitely not mycoplasma. Possibly, it's debris. I would suggest to spin cells in low rpm to get rid of the dead cells.
Hi Mohammad
Could these just be dead cells? I also grow a cancerous cell line and when the cells die they turn into very small round cells which look nothing like the adherent ones. I have also one had a bacterial contamination in them. It's very clear to tell when it is contaminated, as you can see rod shaped cells vibrating in the media. Perhaps just try filling a flask with empty media and having a look at that under the microscope to see if these dots are found in the media.
Post a picture. My cancer cells are getting a dot like contamination also. I am confused what is going on?
it's contamination. you might have frozen them contaminated, thats y when u thaw cells, u can still see them. DMSO in freezing medium MAY eliminate them, but all the times.
Hi,
Thank you for all your messages
I do face similar situation with Vero cells.
So in the end could you find the reason of these black dots like particles?
Thank you very much for your time and consideration.
Best Regards
I have this problem with MCF-7 , I thaw them and after 24h there was some dark dots and after 48h they became more. Some of them are attached to the cell surface.
The black swimming dots in your cell culture are Achromobacter. It is a new discovered contaminant in cell cultures. It called the attention of many scientiscts and there is even a paper about this: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2849847/
Hope this answers your question. Let's see if we can get rid of these "new" bacteria from the cultures :-)
They are definetively not cell debris!!!
Good luck
Josema
Dot like particles are contaminations, as they keep on growing and if you keep culture for longer, they occupy most of the plate space. So if you have some other source of the cell, just discard this, otherwise you have to treat them with combination antibiotic therapy.
I have been facing one more issue. The cells seems to be bursting after passage number 5 or 6..till then they seem to be growing fine..there seems to be a lot of dead cells that stick to the growing cells and also they float in the media. Is it due to phage contamination that the HeLa cells are bursting? Should i reduce or completely remove antibiotic from the growth media. currently i use 1% penstrep solution,10% FBS for culturing HeLa in 5%Co2 incubator. Please advise.
Hi Mohammad Ishaq
Did you manage to get rid of those dot particles from your culture. If Yes then do let me know how??? I seem to get those dots in my HEK-293 cells also.
Hi Priya.
Yes I got rid of those dots but I can not specify exactly what was the source of these dots. I discarded all the cells in culture, and media, FBS, .... Almost after10 days I started with a new culture in a separate hood . The hood I used was from a different lab and I maintained the cells in their CO2 incubator as well. I also used 2 fold Conc of antibiotics. Once the cells were ready, i divided them in two flasks and brought one in my lab. Cells from my lab as well as from other lab were then without any dots. After that I have never encountered the same problem again.
Good luck...........
We are also facing the similar problem in our lab. I don't think these black particles are some sort of cell debris or any other inanimate things - It seems that they are living contaminants, as they exhibit a specific swimming pattern and also increases in number as the culture duration increases. It's a good indication of bad laboratory practices.
We are also facing the same problem. we used the combination of antibiotics like ciprofloxacin and piperacillin along with usual streptomycin-penicillin antibiotic. But there is no change in the growth of dot-like particles.
Hi
These particles were persisting even after reviving the frozen cells. I think it may be some invading microbes? I can reduce their number by washing the cells with 1x trypsin - EDTA solution regularly. I suspect these dot-like particles may interfere the results of protein estimation, western blotting, immunofluorescence etc. I hope this can be removed only by using new cell lines after discarding all the older cells in a newly fumigated hood and culture room and the incubators.
The particles you see might just be microvesicles generated by the cells in culture. The movement is Brownian motion. If it were bacterial or fungal infection, the culture wouldn't have survived. Mycoplasma is different and doesn't necessarily cause cells to produce dots and may require PCR for detection.
I had faced this same problem also. But in my case, what eventually was discovered was that we were using expired DMEM from Cellclone. The vendor who supplied the reagent forged the expiry date and gave us the media. When we used GIBCO DMEM, the cells were growing healthy, no dot particles and the rate at which they grew also changed. In GIBCO DMEM HeLa cells grew doubly fast as compared to Cell Clone media( not the expired one, but the proper one). It varies from company to company. local brands are cheap but do not have effective QC checks. I hope this helps.
It may or may not be contamination. I too faced the problem, but in order to assure it I further went for MTT assay just to check whether they are Live bodies or not.
To my surprise I found they didnt stained (formazen crystal formation) on analysis under microscope. So in order to overcome this unknown hurdle, iI started doing passaging a lot of my T25 flask, until I get minimum of those black dots within the culture. With the time the spots disappeared and I got reproducible results in my MTT assay.
During first hours/ days after thawing the apoptotic cells debris will be formed. The magnification is to low to distinguish but this is normal thing when putting cells to culture from frozen vial. This do not exclude contamination. Check in 3-4 days. Have fun with Your cultures!
HI
You have got bacterial contamination in your cultures. Just thaw a new stock and then if you observe the same problem then revive a new stock of some old date and discard all stocks of the date in which you are getting contamination.
Since it's a cell line so there is no need to bother much. Simply get a new stock and revive it properly. And maintain personal hyeigene...Simultaneously u can also run an other cell line parallely and you will get to know wheather the contamination is inherent in cell line or due to some other source (as both will get contaminated if contam source is something else)
hope this helps
Hi Mohammad,
Two weeks ago, I faced similar issues when I was culturing DLD1 cells in the T75 flask. Initially, I thought it was some kind of contaminant (e.g. yeast) or death cell debris because the size of the dark particles is much smaller than the DLD1 cells and the dark particles were not floating in the medium. The strange thing is even with the presence of these black particles; my cells were growing and proliferating really well. Later I cleaned our equipments and prepared fresh medium and PBS solution, but the condition did not improve even after these approaches. Thankfully, my colleagues helped me to troubleshoot this issue. It turns out the dark particles came from the exterior of the flask. We targeted the source of the dark dots actually came from the gloves. Some powders were actually coming off the glove when we spray those with ethanol. My suggestion is first to wipe the bottom of the flask with kimwipe and see if the dark dots are gone or not.
Hope this is helpful for your work and good luck,
Alexx
I am also experiencing similar problem with my cells. I will try out some of the suggestions one at a time to get rid of these black dots.
Many thanks for your useful suggestions
good, it is a long story. I suggest a heavy combination antibiotic treatment. You loose many of the cells, but the remaining will get OK once you bring them back to normal antibiotic treatment/
Hi Mohammad,
Was your problem solved? I am having the same problem with MCF7 and MB231.
Thanks
Shamim
I would like to express my opinion.
I have compared quantitatively virus contents specific to the cancer cell.
Hepatoma HepG2 (cultured without fucoidan) has uniquely
Polyprotein (GB virus C/GBV-HGV/GBV-C) at 15.4, Genome polyprotein (Human poliovirus/PV) at 5.1, Hemagglutinin-neuraminidase (Newcastle disease virus/NDV) at 1.6, and Envelope glycoprotein gp95 (Rous sarcoma virus/RSV) at 0.5 μg/mg of cell protein, respectively.
These vira are not present in fetal hepatocyte Hc and healed hepatocyte HepG2 (please see file; HepG2 fucoidan).
Therefore, your phenomena observed in some cancer cell-lines may be caused by membrane glycoproteins of either GVB-C, PV, NDV, or RSV.
Even i am currently facing the same problem with my cell line of Huh7.5. and i am not able to rectify at all. once i thaw the cells it looks very healthy for 1 passage and after 2nd passage, before the cells could grow, the background with dirty dots start appearing not letting the cells to grow properly n eventually cells looks so stress that we will not be able to differentiate the cells with background. I have been facing the problem for more than a month. and i am scared to thaw any new cells. Can it be due to the plate, or CO2 supply o any other reasons.? Please help me troubleshoot this problem..
Hi Aunji, from your description this does not sound like the same problem as the dots described in the original question. I think you may have a contamination but I can't be sure without looking at an image. It would be helpful if you post an image if you have one, especially at the later stage where you said you couldn't identify your cells from debris. The source of contamination could be your media or other consumables but also possible that the contaminant was frozen with your cells at a low dose and grew later. Maybe try to culture your cells again with fresh sterile consumables and aseptic techniques. If this improves your culture, then the source would have been your consumables. If not, then it is in the frozen stock.
I aslo meet the same problem, but my superviser does not agree with me ,she think it is normal condition. But for me ,I feel these black dots are inhibiting the growth of my stem cell. I feel very anxiety about it. I also told my boss, but he did not say anything about it.
Hi ZHANG,
I hope you have already checked for the bacterial and other contaminations.
If you are culturing stem cells in B27 media and the cells grow in spheroids, then you will always a bit of particles. Actually these are dead cells. So go ahead with the experiments as ur PIs know this very well.
Good Luck
Best
Ishaq
Thank you for ur answer,I notice these dots dance and swim!no bacteria !
I recomend to set up the microscope to darkfield, 20 X magn or more . Then You can seee better what You have.
Hi,
Try adding bleach to your culture flask, and observe under microscope. You will see lot of black particles coming out of cells. It is just the debris or the excretion of the cells under stress condition. Try changing your culture flask to Nunc or eppendoff.
Hope this helps.
Thanks
Nisha
I also have the same problem with PUREC cells. Its appear and continue propagation after 2-3 passage. I am also still dont know what is the problem with my culture.
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