I am trying to separate and culture human monocytes.But I still could find any macrophages/monocytes in the flask after 23 days culturing.
My procedure is :
1 Use Ficoll paque to separate lymphocytes and monocytes from blood.
2 by incubating with RPMI 1640 for 4 hours, remove lymphocytes from the suspension, those cells attached on flask wall were considered monocytes.
3 culture the monocytes in 37C 5% CO2 condition, changed medium 3-4 days.
For RPMI 1640 type, I used the formula with L-glutamine and added 2 g sodium carbonate in 1 Liter.
I am not sure if the monocytes can split or not?
How many days take them to become as big as 50 um?
What's the problem with the procedure?
I highly appreciate your help.
University of California-santa barbara