Problems with J774A.1 cells

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mlfraga
mlfraga's picture
Problems with J774A.1 cells

Hi,
 
we have had problems with J774A.1 for over a year now and we don't now what else to try. They seem to grow fine in flasks but when we plate them into plates to do transfections, every now and then, randomly, they will die. One day after plating, even before transfecting, cells in the wells will be devastated, specially in the middle of the well. This always seems to happen after passage into plates, but not every time we do it. Also, it doesn't happen in 100% of the wells, but in most of them. We can go for weeks no problem, and then, one day, for no apparent reason, the "effect" will happen again.
 
Things we have noted and tried/checked with no sucess:

  • our media is never older than a week
  • our cells are always low passage (mainly because everytime this "effect" occurs we thaw a new vial and start from scratch
  • we are carefull with trypsinization time and we always trypsinize+scrape to detach cells as they are so highly adherent
  • we have tried different plastics
  • we have tried different CO2
  • we have tried culturing the cells in 20% FCS instead of the usual 10%
  • we have ordered new vials from ATCC

This is tremendously inconvenient as we plan weeks of experiments but we can never be sure if the cells will do ok or if all the plates we just prepared will be wiped out the next morning.
 
I would deeply appreciate any comments or suggestions or to hear of anyone who might have  had a similar problem and was successful solving it.
 
Also, does anyone have experience with any of the other J774 cell lines? I found another thread asking about these other cell lines but not much discussion followed so I wonder if anyone has more current knowledge about the issue.
 
Thanks
 
Marta

Jason King
Jason King's picture
I know this might sound

I know this might sound stupid, but are you sure you didn't use un-treated plates by mistake? Are the plates you refer to 10cm petri dishes? (as used for pouring LB plates etc). We have had this problem in the past. The student that stocked the tissue culture lab grabbed a number of packs of petri dishes and amongst them were some "Sterilin" plates that we usually only use for bacterial work.
The fact that the cells in the middle of the plate look worse could be because this part of the plate is raised slightly and as a consequence contains fewer cells and less medium (depth).
 
Trypsinizing AND scraping shouldn't really be neccesary. I used to wash the cells twice with PBS (no Ca2+) then add the trypsin and watch under the microscope until the cells began to detach. Then add fresh medium to stop the trypsin and resuspend the cells.

heehawmcduff
heehawmcduff's picture
I have used both the J774.2

I have used both the J774.2 and J774.A1 cell lines and there are certainly differences between them.
I would certainly recommend switching to the J774.2 line as they are more robust, suffer from less TC necrosis, are easier to passage and recover quicker from liquid N2.  They also display higher phagocytic rates, and chemotactic potential
However, when comparing chemotaxis/phago to primary isolated alveolar macrophages, the J774.A1 cells are more similar.
As for passaging, I never used trypsin with these cells.  Simply scraped them off with a Sterilin cell scraper.
Just grasping at straws here, but the media you are using is RPMI 1640 (works better than DMEM for these cells) + 10% FCS + 1% fresh L-glutamine?

mlfraga
mlfraga's picture
Hi again,

Hi again,
 
thanks for the feedback so long. A few comments:
 
Pavorman: yes, our plates are treated. We are currently using Corning Cell Bind plates. We might change to a different kind of plates that don't have this cell bind treatment (though they would still be cell culture ready) to facilitate detachment of cells because with these one, we really do need to trypsinize and scrape the cells in order to passage.
 
heehawmcduff: Thanks for the info. We just ordered J774.2 a few days ago so we'll see if we get luckier with them. Regarding the media, we are indeed using RPMI as both ATCC and DMSZ (where we have purchased J774.A1 cells) recommend it.  We might give DMEM a try as you suggest. Thanks for the tip.
 
Marta
 

Jason King
Jason King's picture
Just to add that some people

Just to add that some people in our department are now using temperature sensitive TC plates which enable them to passage macrophages more gently. I don't remember the supplier's details (any ideas HeeHaw?) - just that the plates are quite new and their supply is not high enough to satisfy current demand (at least in the UK).

heehawmcduff
heehawmcduff's picture
I've never actually heard of

I've never actually heard of temp-sensitive tissue culture plastic but if you do find out any more about it, i.e. suppliers details could you please post.   It may be good for primary airway passaging too.
 

mlfraga
mlfraga's picture
I just bumped into those

I just bumped into those temperature-responsive cell culture plates you mentioned. They sound pretty cool indeed.
 
If anyone is interested in checking them out:
 
http://www.nuncbrand.com/us/page.aspx?ID=11850
 
http://www.nuncbrand.com/us/frame.aspx?ID=11867

ykisselova
ykisselova's picture
Dear Marta,

Dear Marta,
try NOT to tripsinize the cells. according to ATCC guidelines they should not be trypsinized!!!!

best regards
Yoana

isantarino
isantarino's picture
Dear Marta,

Dear Marta,

I just had found these comments here as I will have to transfect J774A.1, and i would like to know if you were able to perform the transfection protocol for this cell line. I know that this post is from years ago, but i got interested in.

Thanks a million!

Stevencd
Stevencd's picture
have you solve your problems?

have you solve your problems? If not, you might find some help of the experts in Creative Bioarray, I also do the cells research  many years.