Cell culturing questions

[att159]

Hi, I'm doing a project regarding the effects of amyloid beta on TNF-a production in mouse macrophages. I have some questions regarding the culturing of j774.2 macrophages:
1) Time to culture? I am receiving them frozen, so how long will it take to get them to a point where they can fill a 96 well plate? I know the doubling time is 1 day, but I haven't been able to find any specialized protocols for mouse macrophages online.
2) Would anyone know of any macrophage culturing protocols online? I have found general cell protocols at Sigma Aldrich and other places, but I need one for macrophages.
3) The facility I am using only has a regular incubator, not a CO2 one like I need. I have emailed another professor (who does have a co2 incubator) about it, but in the event that it doesn't work out, I need to have a backup plan. A person at Schmahl said that for bacteria, they've done this thing with co2 gas packs to create a c02 environment. would this work for cell culturing also? Have you ever heard of something like this? It seems doubtful to me, but I've never done cell culturing before.

I would really appreciate any help you could give me! Thank you so much!

[MelissaB]

Hi,

I'm afraid I don't know anything about culturing macrophages; I've asked the other experts to chime in if they do. But, have you tried asking your mentor (or a grad student or post-doc in their lab) these questions? They may know a lot more about the system you're working with and have better advice for you than we will have.

[deleted-71607]

I agree with the other expert's sugguestion of speaking with your teacher or other researchers in your lab. In addition, you can look up papers where they used J744.2 macrophages. Then, you can e-mail the corresponding authors of these papers to ask them for suggested protocols. You can also ask them their suggestions for culturing the cells without a CO2 incubator. I hope this helps.

CLKline

[att159]

Thank you both so much for your answers. I really appreciate it.

[MaryB]

Hello,

I just wanted to check and see how things were going with your J774 cells. I routinely work with mouse macrophages and have attached a link to an optimized protocol for another murine macrophage cell line (RAW 264.7) that should work for J774 cells also - see below. Sigma protocols are generally pretty good too. You will need a CO2 incubator to culture/maintain your macrophages - I have not heard of using gas packs for cell culture.

http://www.lonzabio.com/fileadmin/group ... V-1021.pdf

I am not sure what point you are at in your project, but feel free to contact me with any questions.

Mary

[att159]

Hello,

I just wanted to check and see how things were going with your J774 cells. I routinely work with mouse macrophages and have attached a link to an optimized protocol for another murine macrophage cell line (RAW 264.7) that should work for J774 cells also - see below. Sigma protocols are generally pretty good too. You will need a CO2 incubator to culture/maintain your macrophages - I have not heard of using gas packs for cell culture.

http://www.lonzabio.com/fileadmin/group ... V-1021.pdf

I am not sure what point you are at in your project, but feel free to contact me with any questions.

Mary

Hi Mary,
Thanks for the response and for the protocol - I'll definitely use it. I know that having a c02 incubator is the best way to culture macrophages, but I emailed the source for the macrophages (HPA cultures) and they recommended (for culture outside of c02 incubator) either HEPES medium or manually gassing the flasks with 5% c02 and then sealing tightly. I will be doing both to be safe.
I'll definitely email you if I have questions - once again, thanks for the link.

Anna