i am culturing human monocyte from pbmc after that sorting with Pan monocyte kit. i add 50ng/ml of MCSF and after 5 days add IL13, IL4 for 24 hr. to polarized them. After that i harvest and freeze them to co-culture with human lung fibroblast.
please explain how to harvest the monocytes because when i harvert and plate them again they never adhere.
Are you differentiating these monocytes to Dendritic cells? GM-CSF and IL-4 can differentiate monocytes into DC, which are non-adherent cells. You don't need to detach these cells; gentle pipetting is good enough to harvest these cells.
i am using GMCSF and il13, il14 to make them adhere once they become polarized i can to co-culture them with human lung fibroblast already seeded. so i have to the monocytes with them so for this i have to detached them. after the don,t attach again
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