Hi Angie,

I've never used J774A.1 but I've had problems in the past with culturing ATCC feeder-free hIPSCs. Turns out they weren't actually feeder-free, I had mixed morphologies as well. The solution to issue #1 may be to simply switch providers/cell lines.

For #2, try accutase. It's gentler than tripsin though with a longer incubation time.

Hope this helps

Hi,

though I did not work with your particular cell line yet, just a few general ideas:

We have cells with morphology similar to those you marked on your picture in virtually all cell lines; I have been explaining them with altered cytogenetics (most probably tetraploids). Take a careful look on the upper left cell: It is binucleated, which also explains the increased size. From my experience, a certain rate of such anormalous cells is unavoidable especially in permanent cell lines. The one in the middle to the left may represent an apoptotic cell, but from that magnification that is just a guess.

Something else to note: Almost all cells in that picture show significant vacuolation. While this may be a characteristic of this particular line, it is in general a frequently observed indicator of cellular stress and / or senescence.

For cell harvesting, for loosely adherent cells you can try using modified PBS (w/o Ca2+/Mg2+) in conjuction with agitation of the cell culture vessel (e.g. hard tapping against the palm of your hand). Alternatively, there are different concentrations / compositions of trypsin, it everything fails, maybe try 0.05% trypsin-EDTA solution to minimize cellular damage.

Kind regards

#2 We've also experienced problems with harvesting, in our case freshly isolated human monocytes and macrophages. We have tried EDTA (on ice or at 37C), Citric saline (on ice or at 37C), Accutase, Lidocaine or Trypsin.

Bouke:

I've heard good things about Accutase, and have seen the lidocaine protocol. I'm no familiar with the citric saline protocol. Which do you think provides the least toxic approach to counting cells, and for any of our teaching assays that involve detatching the cells? (And for plate seeding purposes of mine as well)?

Keagan: Where is a better source for ordering cells?

Patrick: I felt that the uppermost cell bi-nucleated as well; I also strongly agree with your comment on the vacuoleation in the cells. But these aren't seen under the normal magnification used for counting cells on the inverted scope. I have a FLoid cell imaging station, which allows these really close up pictures, so you can actually see all these "little things" that are going on.I appreciate your insight!

I will try your suggestions as far as far as detatching the cells tomorrow. many thanks!

Citric saline: 135 mM KCl, 15 mM NaCitrate.

Some claim that citric saline is less harsh than EDTA or trypsin and that the cells reattach well. You'll have to try a few methods for your cells.

Hi Angie,

I work with J774A1 macrophages from ATCC too, and I must tell you is quite a tricky line, because of the many variations they experiment during cultivation. They grow very quickly and it is tough at the beginning to control it (specially when seeding them in 96 well plates).

I agree with other comments, you have much vacuolation in your cells, a sign of cellular stress. Maybe it is the FBS you use or the kind of media (I use DMEM high glucose from Sigma), try to check this. Regarding cell counting we have the same problem, you need to be very careful and try to scrape the surface of the flask with solid movements and not to do it too roughly (By doing this properly you will achieve 75-80% survivability). It's worth mentioning that these cells, in particular, doesn't tolerate trypsin very well, besides they adhere very strongly to the flask surface so scraping is highly recommended to detach them successfully.

I attach a picture of our cells for you to check how they look.

Hope it helps!

Jose,

Thank you so much for your comments!!! I have noticed that the cells do not do well in any of the mediums I've tried except for the ATCC media. I haven't tried SIgma's media yet, but have tried Corning, Hyclone, and Gibco. I switched to flasks, and the cells seem to have recovered nicely. Like you, I have noticed that they appear to grow very quickly, and require subculturing every 2 days. I just split them yesterday, and can already tell that they will need to be split again tomorrow. Have you tried using Accutase or the citric saline for detatching the cells? I've attached an image of the cells after I recovered them and switched to flasks instead of plates.

Hi Angie, Hi all,

very interesting discussion.

I have a suggestion that might be very naïve, not having properly worked with macrophage cell lines myself before.

How about trying to cultivate the cells on untreated plastic ware?

like the bacteriology dishes for example? Ususally regular adherent cell lines (not macrophages or APCs) would grow in suspension. Your macrophages might attach but also detach much easier.

I'd be interested to know if this helps, of course if you see that it's worth a try.

Good luck

Georges

Georges - I'd heard of doing that, and plan to try that out next week. What I have figured out is that this cell line prefers to be cultured in a 75 ml tissue culture treated flask. ALSO - when passaging the cells, I remove all but 10 ml of media, then gently scrape them, and pipet up and down to break any cell clumps. Then I split them at a 1:10 ratio into a new flask (I put 2 ml of the cell suspension from the old flask into 18 - 20 ml of fresh DMEM with FBS, Pen/step, and L-glutamine.) This ratio is perfect for seeding 6-well dishes for a 2-day assay, where I will seed the wells on Monday, treat them with whatever chemical I'm looking at on Tuesday, and then Wednesday I can do whichever assay I've chosen. Thank you so much for your suggestion!!!

Hi Angie,

We use a different protocol using RPMI and 2-Mercaptoethanol, 20% FBS for the cell line. After 3 days in incubation, I got my desired cells (P6-P7). I couldn't find any morphological alterations in the cells. What is the passage number for your cells?

What does the 2-mercaptoethanol do? My cells ended up being fine after about the 4th or 5th passage. But in the beginning, they have lots of vacuoles.

Mercaptoethanol is disulfide bond reducing agent which is added to reduce oxidative stress (like an antioxidant) developing in few cells. For us, macrophages tend to attach a lot in clumps/nets and cannot reduce this stress in culture. May be the reason why you are getting vacuoles (may be of lysosomal origin) which is a sign of stress in cells or accumulation of ROS.

Souptik Basu , can you please tell me the percentage of 2 mercaptoethanol you use in RPMI media preparation for J774 cell lines.

Souptik Basu I have the same question as Swetapadma Majhi . I would like to try adding the 2-Mercaptoethanol

Dear Swetapadma Majhi and Ahmed Eid RadwanI used these components for making the media: For 500 ml, 1. RPMI 1640 minus L-glutamine (Gibco 21870076): 417.5 ml 2. Sodium pyruvate 100 mM 100 X(Gibco 11360070): 5 ml 3. MEM NEAA 100X (gibco 11140050): 5 ml 4. Glutamax I 100X (Gibco 35050061): 5 ml 5. Pen/Strep solution 100X (Gibco 15140122): 5 ml 6. HEPES Buffer (Gibco 15630080): 12.5 ml 7. 2-mercaptoethanol 100X (Gibco 21985023): 4 ul 7. Fetal Bovine Serum (Corning): 50ml

Souptik Basu Thank you very much, this is very helpful.

Thank you Souptik Basu .

:) Namaste.