Hello,
I am trying to utilize NR8383 cells, and need them to grow in attachment. The NR8383 cell line is partially suspended and partially attached when it is grown. It has been suggested to culture only the adherant cells, however it seems there are far less adherent cells.
Does anyone know how to successfully grow adherent NR8383 cells? I appreciate any thoughts and ideas.
I have seen different medias used, and I wonder if this could play a role in their attachment. We use Ham's F12K supp with 2 mM L-glutamine, 15% heat inactivated FBS, and 1% pen-strep.
Is there any literature about the optimization of these cells?
Thank you for your help.
I do not know the cell line, but you may try "coating" the cell culture flask with some extracellular matrix proteins to get more or better adhering of your cells: for example fibronectin, not sure if laminin and others may also work. There should be also some cell culture flasks on the market which may offer specifically "charged" surfaces for better attachment, but again, I do not know if this may work with your cells. As a final consideration, if anything fails, you may also think about using some other cells as base, for example, endothelial cells to which the macrophages may adhere better.
Not with this cell line, but I can vouch for poly-D-Lysine coating as it works wonders with several other lines I tried it with.
Yes, poly-d-lysine usually works for all cells; any such attachment may affect the physiology, activation of an immune cell a lot, but this depends on what you want to investigate. So, Poly-D-Lysine may work best for pure attachment, but may also stick the cells too much to the surface. It may be worth a try, perhaps compared to others. Any selection for the attaching cells may of course also change some other characteristics.
Your FBS content is too high. They are less adherent when the total protein in the media is high.
Thank you for the input thus far, I don't think anyone has hit the nail on the head yet. The media + FBS is based on manufacture protocol (see attached link).
Thanks for the ideas
http://www.atcc.org/Products/All/crl-2192.aspx#culturemethod
15% FBS appears to be high for other cell cultures, but I've seen on the official sites that this is recommended to be that high. But the cells may just grow that way - although each batch of FBS might be different to cells. Since I do not know how you will proceed with the adhering cells - do you need them alive for further processing - or could you include a gentle washing step and then fix with either glutaraldehyde or paraformaldehyde ? that may stick the cells better to the surface.
I am currently starting to work with NR8383 cell line (unfrozen the ATCC vial on Friday) and I'm searching for some help with the harvesting/flask change protocol. I tried to check cell density and tried to do Antje advice putting the flask 10 min at room temperature but it did not worked. Cells remained attached to T-flask, maybe because I'm using adherence treated T-flasks. Should I use suspension cell culture T-flasks? Do you have some advice/protocol that can share with me? I would be really grateful. I'm not knowing how will I harvest the cells from the flask to count it and make the experiments with the adherent ones. I think I'm having the opposite problem from Trevor... in my case I have more then 50% adherent cells. I want to pass it to a new/bigger flask to have enough to freeze some stocks but don't know how or if it should be different to freeze adherent or floating ones or should I mix it both?
Can't you use a (plasic) cell-scraper (if I remember right, it is also called policeman) to remove and harvest your cells?
Hey Susana,
It is reccomended by the manufacturer to use a cell scraper as Dr. Eibl mentioned. I would then reccomend pipetting the cell suspension up/down 20x to break up any clumps of cells. You should be able to count the cells and then reseed flasks after this step.
What type of flask are you using? Collagen coated?
Good luck!
Hey Trevor, Thank you.
Yes I did it and change it from a small to a bigger flask but from readings some persons mention to only reseed new flasks with the floating ones. Do you use both? Next week I would like to freeze some -80ºC stocks of it do you recommend me to use both or just the suspension ones? I'm using flasks with adherence treatment but not collagen ones.
For my application I want the adherent cells, so I rinse the flask 3x with media or PBS then use the cell scraper to remove the attached cells. If you are okay with a mixed stock keep both attached and suspended cells, it really just depends on your application.
Yes for my application I will use adherents too but for now the first goal is to make some stocks of this cell line. Have you done -80ºC stocks already? How do you prepare it?
Susana here is the standard operating procedure from our lab. I hope it helps.
Thank you Trevor, It will help a lot.
So you only use the adherent ones even for stocks. I had read that for experiments normally the adherents was the ones used but for stocks and reseed in the new flasks I was wondering which ones to use.
You can freeze two batches and figure out for yourself which ones are more viable after thawing. Freeze the suspended and then freeze the adherent separate. I also recommend only freezing half of your cells, so that way if something goes wrong during freezing, you have cells to continue to culture and try to freeze the next week.
yes I have 4 t-flasks growing and planning to freeze 2 of it tomorrow and check viability and only latter on decide what to do with the others, for other cell lines I have already follow that protocol of keep some cells "alive" until check if stocks are fine. I think I will follow your advice and in 1 flask freeze only adherent cells and for the other both and check which one work better.
Once again thank you Trevor
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