We are experienced lately some problems with primary cells.
We could see (even in 10x magnification) tiny bright particles around the addhered cells.
There is no macroscopic sign of bacterial/fungal contamination (turbidity and pH) and the microbiological assay did not identify consumption of O2 or CO2.
We are used to use cell culture media with Pen-Strep.
Has anyone experienced this before?
If there is no apparent replication of the contaminating particles, then you might suspect mycoplasma. Also, the particles outside the cells are all different sizes. Usually, with bacterial or fungal infections, you'll have a large number of particles that are the same size and often clustered or in strings. There is a lot of debris in the image which could be from cell death or some part of your handling protocol. Try looking at the particles with a higher mag to see if they are crystalline. Check for mycoplasma with DAPI or a mycoplasma detection kit.
If there is no apparent replication of the contaminating particles, then you might suspect mycoplasma. Also, the particles outside the cells are all different sizes. Usually, with bacterial or fungal infections, you'll have a large number of particles that are the same size and often clustered or in strings. There is a lot of debris in the image which could be from cell death or some part of your handling protocol. Try looking at the particles with a higher mag to see if they are crystalline. Check for mycoplasma with DAPI or a mycoplasma detection kit.
Looks like it could be myco, test for that. There is a lot of debris in the pic, could be because of cell death or media is not filtered properly..
Those look like dead cells to me, which is common in primary cell cultures. You should definitely check for mycoplasma though as it is highly prevalent in the oral cavity. I've used the R&D kit before and it seems to work well. Alternatively there are companies that will test your cultures for you(I would recommend this if you only have 1 or 2 samples)..
My answer is similar to the Ron's. According to your description, mycoplasma contamination maybe the root of your crisis. You would solve it with some myco scavenging agent, for example, the ciprofloxacin solution.
Hello Bruna
First at all, you should do mycoplasma test. However, I think that your cells are in stress stage. I advice you do a citometry study with anexina V-propidium iodure to know the integrity of membrane (early apoptosis). In addition, analysis of BAX/BCL2 by PCR is indicated whether the cell are in mitocondrial stress and it is related with intrinsic pathway of apoptosis activation.
Best regards
Looks like a yeast contamination, which will increase in a day or two. You have to destroy the affected cultures with bleach. Incubate a sample of your fresh media for 24 hours without cells to see if it is contaminated. If it is throw it away. Make sure instruments, TC hood, dishes are all sterile. Start new cultures.
Hi! I agree with Oscar, we also work with mesenchymal cells from dental pulp and sometimes our cells look like your pic. We tested for myco by PCR and was negative the resoult, then we though on stress...this cells are used to grow under hypoxic conditions and we put a plate under 1% O2 for 3-4 h, then we look the cells and all the dark points and tiny bright particles disappeared. After 3-4h of normal conditions of culture appear againg. For this reason I though on stress...but I dont know more. We culture it with DMEM low glucose, FBS, and P/S.
Thank you very much for all the help.
We made myco test by PCR and was negative.
Can I make an homemade hypoxia chamber to test the cell stress?
home made hypoxic chamber...ehhh...if you have 2 incubators put one at 1% of O2 conecting a N2 cylinder to occupy the volume of oxygen inside the incubator. If is not posible you also can try treating a small sample with cobalt chloride, it mimicrys hypoxic conditions but you will be using a chemical compound...
I personally think you should throw your lines away, clean the filters of your fume hoods and sterilize the hood again. Open all new media and media additives. Costly, but better in long run.
Likely you have other stocks of cells (early/ pre-infection) frozen back, or can ask collaborators for replacements. You can filter the lines, and use Fungin etc, but once you have an infection it is difficult to entirely clean it out. You also risk having un-reliable experiments and faulty data later on.
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