Dear Colleague,

I agree. Often, when cultivated in 96 well plates, the number of cells per well is to low and doesn't allow the cell to grow again, and they die (and it's right that some factor secreted by cell are necessary for the growht of the others);

I think you have to calculate a number of cells in each well, in order not to be under about 200.000 cells per milliliters (there is a threshold of concentration under which start again of growth is more diffcult; especially after the "cell stress" due to sub-cutlture.

Finally, the sign you observed (little bubbles) could be a non specific one of beginning of death, of perhaps contamination by mycoplasma; but you would have observed in this case with you original culture (but you could test with specific mycoplasma detection kits).

Regards

Didier J

Dear Colleague,

I agree. Often, when cultivated in 96 well plates, the number of cells per well is to low and doesn't allow the cell to grow again, and they die (and it's right that some factor secreted by cell are necessary for the growht of the others);

I think you have to calculate a number of cells in each well, in order not to be under about 200.000 cells per milliliters (there is a threshold of concentration under which start again of growth is more diffcult; especially after the "cell stress" due to sub-cutlture.

Finally, the sign you observed (little bubbles) could be a non specific one of beginning of death, of perhaps contamination by mycoplasma; but you would have observed in this case with you original culture (but you could test with specific mycoplasma detection kits).

Regards

Didier J

Did you trie change de medium? After some days you need add more trophic factor. They degrade over time, and maybe that is happening with your culture!

Which factor have you used?

Try with the different factors in electroporation and the media components that you use may have some default

Try using spent medium (50% medium from a culture 3-5 days old with 50% new medium). I am assuming your cells require serum and are not PFM. If PFM, look for commercial medium that can support single cell growth.

Dave

I am agree to David petch

... also, Karina, while striving to for what David Petch and other colleagues suggest, remember to check your cells after electroporation to avoid possible membrane fusions. Membrane fusion may create cross-talks between your ES cells. These maybe the dying ones.

Gilbert.

How depressing! I'd try glass plates, if possible. Treated/coated. +/- trophic factors, +/- vary the plating density (sometimes they like to share trophic factors with each other, maybe?). Good luck! This is a PR bulletin from Nunc, but may be helpful? http://www.well.ox.ac.uk/cytogenetics/downloads/nunc%20coverslip%20chamber.pdf [PDF]

Hey all,

thanks for you support!

At the end of the last year we had two successful targetings with the same cells I am using now. I do all the steps like they did, so it's relly a mystery to me why it's not working...

We also thought that it might be the plates as I used plates from a new batch, but when I seeded clones from a colleague they grew just fine. We also changed our pipette tips so I thought that this could be the reason, I normally pick them with these tips ans also for resuspending the cells after splitting the cells have contact with the tips. I will start the next try on Monday and use the old tips (I fortunately still found some boxes in our lab).

The medium I used was used in parallel for cultivating other ES cell clones, so it seems to be fine and in ES cell culture we change it every 24h...

As I am working with the same protocol as my colleagues it's quite strange that it is no working. Can sifferent KO-construncts lead to so different needs for the ES cells?

Cross fingers for my next targeting, I will keep you updated...

A last think, Karina: how many passage of your cells since the beginning of culture and sub-culuture, and do you use regularly new "fresh" aliquot who where frozen at the beginning of the culutre when you received them ?(or isolated them; I don't know your context); or are these cultures provided to you from a commercial source ?

Why these question: I thiknk you are probably used with the fact that , even immortalized, cells in culture can "loss" their capability to divide and continue to growth after a certain time, without especially precise reasons (programmed cell death even with immortalized ones ?).

Best regards

Didier

Reduce cell density, Check Osmolarity during culture in 96 well plates. Follow according to Didier suggestion.

regards

soosai

I think it is co-related with the co-construct (I do not know what type of construct exactly that you used?) you mentioned that you did by electroporation

if not it will be the type of cells may be contaminated by mycoplasma during the transformation

If your culturing conditions are the same as prior targetings, as you have suggested, I would look at the batch of plates. It is not uncommon for some plates or batches of plates to be toxic.

If the problem is occurring around the edges of the plate then there is a humidity or CO2 problem and lastly possibly contamination.

I think it's due to contamination

You can add some FBS (about 5%)

well ,,, i dont have any experience about it but... it can be said may be electeric current is causing DNA damage

yea, it sounds like it's contaminated, but then I wonder why the cells always die after the same step (first splitting after picking, this happened three times!!!). And all other solutions/media have also been used by others, so they should be fine...

I am starting the next try... let's see what happens!

u should do culture in T-25 CULTURE PLATE, after then when it is sured that there is no any type of morphological changes, then you should seed in 96 well plate....

even it was same as earlier then u go through new ES cell revive.

some suggestions from a chemist:

what about using positive and negative controls?! That is:

A) use some wells with not-electroporated cells (negative control), if they also die, it has nothing to do with electroporation

B) use some wells for growing electroporated cells with a "harmless"/ proven/ working KO construct (positive control). If the show the desired growth, it has to do with your KO construct...

In this way, you can check out all those assumptions by experiment, and you will not be damned to try the same thing over and over until some time it worked by chance...

I agree with using always positive and negative control for any experiment this is one of basic standardization of assays

but I insist that this syndrome is may related to the electroporation tools and handing

Well, I don't have any experience about it but I think its due to contamination.

Dr.Kulkarni T.R.

Professor in pharmacology, B.V.D.U.M.C.Pune.

Dear Colleagues,

If really the problem could be due to contaminations, and with the cell signs obsreved perhaps mycoplasma, evidently prevention in cultures in general is the best way to avoid it; but this sort of germs contaminating a culture is resistant, intracellular and can bypass simple desinfection.

And when installed, if precious cuture needed to be conserved, difficult to treat (associations of drugs) and long.

So, if the cultures can be done again, all remove them and perform a hard decontamination of environment.

Best regards

Didier

PS see linked a presentation by an industrial on this problem

Please folks, what the hell should we do with a comment like "I don't have any experience about it but I think its due to contamination". As long as the experiment wasn't set up with proper controls checking for the main failure modes, it's completely useless to reason about what to do against contamination, right?

Sorry for this, I just read the article from the link below:

http://pipeline.corante.com/archives/2012/03/29/sloppy_science.php

Dear Colleagues,

Obviously, when we performed experiment with all sterile conditions, some personal experience in cell culture and what is necessitating for , we could hope to have non contamination. But, with a ten year experience in the filed (in the past), I think it's cannot be as simple, and for different reasons:

-first, have we always the material possibility to be sure to have no contaminant: evidently, in the past, in bacteriology or research, we worked only without laminary flow and use of bec bunzen and flam during experiement. In these condtions; which evaluation for contamination rates ? difficult to say because when it was the case, we troughed out the cultures, Then new conditions (laminary flow ie). New rate, lower, but always distinction: which conditions in research ? for simple culture , not use of P4 category security laboratory, which is the case in high virulent pathogen studies. But at present, for all researchers, do you use all, when it's not in human genetic diagnosis where it's obligatory by laws, 3 different rooms for pre-PCR (extraction + mix solution preparation) and PCR, post-PCR ... probably not (confirm me ) for resarch cell cultures ..

- second: the rate of conatmination seems to be correlated not only with material conditions, way of manipulator manipulation (and his experience) , but also with the number of different cell culture made in a same palce at the same time, number and circulating person, cultivating in a same place. In a ten yearperiod where I was alone, I had only one general contamination (necessitating desinfection of all materials) and five cultures put in the trash. When later, more people worked with me on different culture, it was more frequent; and often simply because we wear and tranport contaminants on us,

- some contaminant as mycoplasma are not like classical bacteria contaminant, which you can "see" in our flasks rapidly, but as virus: "sneaky", silencious, not directly visible but acting on cells with stop of growth and damages on a long period.

So, finally, I think we cannot "accuse" other colleagues of bad manipulations, lack of experiment (even if it's often a cause of possible contamination), before having analyzed all possibility. And the signs described by our colleague, as little bubbles in cytoplasma of cells, stop of growth without necessary immediatly die are often met in mycoplasma contaminations.

As said by Jesus in Bible "let the one who has never be a sinner to thraw the first stone " :-)

Best regards to all.

Didier J

It is sometimes necessary to have more time to understand some trivial things.

Hi Karina, I'm currently facing the same problem with growing hESC that has been transfected by electroporation in 96 well plates. They recover really well but when I start picking them into 96 well plates they don't grow. Have you by any chance figured out what went wrong with your experiment? It would be really helpful for me if you can share some ideas from your experience. Thanks in advance!

Mycoplasma contamination is often undetected, unless routinely tested by PCR with known mycoplasma DNA sequnces. Kits for such testing are commercially available. However best practice is to culture your cell lines without antibiotics so that any bacterial or mycoidial infection becomes rapidly visible, allowing one to correct one's technique. With good technique, mycoplasma infections then become much rarer.

Good Luck.