Dear All,
I am isolating monocytes from Bone Marrow of mouse then differentiate them in to macrophages. For my experiments, I need to culture them in suspension. Anybody have any idea how would that be possible?
Regards,
Sami
i have worked on it.. I'll send you the protocol. Its in my thesis too.
Its very easy.
1mRBMI+20%FBS+10 µl antibiotic (penicillin+streptomycin) incubated in 5-10Co2 exchange your complete media after 24hrs incubation.
Simple: RPMI-160+10% heat inactivated FBS, 2-ME, glutamine +100 ng/mL mouse CSF-1. Seed cells at 3x10e5/mL, for Seven days of culture and you will have a plate full of macrophage. Note, they will be stuck to the plate. This requires 0.5-1 mM EDTA in PBS for 10-15 vminutes to help remove them. Trypsin will not work. Good luck, Ken
Thanks Kenneth for the detail answer. I do the same but I was asking if there is some method by which I can keep the cells in suspension for a day. I dont want them to adhere and I would treat them with some particles in suspension.
secondly, you mentioned trypsin would not work, but I usually trypsinize them. what happens if I trypsinize them??
Thanks
I understand. Use Corning Costar low adhesion plates. They are great for culturing mouse macrophage. They just don't stick to them. However, don't use them for human macrophage. The low-adhesion plates come in all sizes (6-well, 24-well 96-well). Trypanization strips many molecules off the cell surface making the cells temporarily less functional. But whatever works. Good luck K
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