Currently I am working on PBMCs-derived monocyte differentiation into macrophages with mcsf (100ng/mL) for 6 days and I give experimental stimulation for 24 hours. My media is composed of RPMI 1640 + 10% AB human serum and 1% Pen/Strep. The experiment is done on non treated 24 well plates at a concentration of 1million/mL where I add 1mL/ well. I change my media on day 3 and day 6. However, I lose a lot of cells upon harvesting.
My questions are:
1) Adding 1million/ well is the right concentration? I have seen forums where people suggest using 500-700*10^5 monocytes/ well.
2) What type of plate works best? Non treated 24 well plates work fine or should I try different plates? Should I collagen coat them?
3) I have seen people first doing the mcsf priming on plastic flasks for 7 days where they change media every 2 days and then they remove/ resuspend cells at equal concentrations and transfer to 24 well plates overnight at 37 degrees +5%CO2 followed by 24 hours of specific stimulation. Do anyone knows if that approach yields better results or working directly on 24 well plates from the start can provide equally good results?
4) For harvesting, I read using enzymatic approaches (trypsin/ accutase) affects receptor expression and recommends using non-enzymatic approaches such as adding cold PBS+2.5mM EDTA on ice for 30 minutes followed by cell scrapping. Does anyone has experience with that approach?
5) Since I am using serum as part of my macrophage media, how necessary is adding MCSF. I have seen forums where has been suggesting not using mcsf and if used they recommend at concentrations up to 50ng/mL. I use 100ng/mL is that affecting my cell survival?
1. Please find attached image for cells number and medium per well volume for cell culture.
2. I use Corning tissue culture plate and it work well. Any plat will work well. You need not to coat with collagen.
3. Working on the same plate for whole experiment will be better as your adherent macophages would be damaged/activated during transfer. You should differentiate cells in the same plate which a to be used for further stimulation. (Make sure to remove all the non adherent cell next day)
4. I need to take RNA and protein from the macrophages so I use scraping but for performing flowcytometry or imaging using accutase will be better option.
5. MCSF will give you better an assured differentiation.
I hope It is helpful.
Hello Juan,
I will try to help with your questions
1) I don`t recommend using 24-well plates to differentiate your cells. Instead of seeding monocytes, start seeding your whole PBMC to a T75 or even 15cm petri dish and after your cells have fully differentiated, then transfer the macrophages to the 24-well plates. However, you will need to seed more then what is recommended by the manufacturer because after differentiation these cells won`t proliferate anymore. So instead of following the manufacturer "seeding density", go for a number closer to the "confluency density" (maybe seed 60% of confluency density because macrophages are big cells and those manufacturer parameters usually use Hela cells).
2) No need for collagen coats. But I strongly recommend using bigger recipients to differentiate your cells. I think 15cm petri dishes work the best. I believe, similarly to what we do to frozen cells, we should never seed cells directly on the plate intended for the experiment.
3) I think my answer on 1) and 2) already covered that.
4)Yes, I use this approach specially for flow cytometry. I remove the media from my macrophage flask and add only cold PBS, leave the flask on ice for 30min-1hour. After that I gently scrape with a soft rubber-tipped scrapper. Works great.
5)I recommend to keep using 100ng/mL of M-CSF. However, I don`t recommend changing the media for the first 7 days. If necessary, add more media but don`t remove the old one (otherwise you will loose cells). Just seed your PBMCs in a big flask with media with CSF. Don`t wash it or change the media for 7 days.
Good luck with your experiments.
Hope that was useful
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