how obtain monocyte from PBMCs and differentiate into macrophage - (Jun/11/2008 )
Hello everyone! I'm new to this forum and i have already a question: i need to obtain monocytes from PBMC and than differentiate them into macrophage, how can i do this?
well, i know the method to obtain PBMC with the Ficoll centrifugation, and to separate monocytes from other cells i allow PBMC to adhere to the bottom of flask with an overnight incubation at 37 °C and 5% CO2.
At the end, i have a lot of cells that are attached to the flask.
What i have to do is to differentiate them into macrophages and than detach the cells for the stimulation: how can i differentiate and detach them?
I've tryed to culture for about 6-7 days with RPMI 1640 + 20% of human serum, changing the medium every 2-3 days but i don't know if this have differentiated them...and than i've tryed to get them from the flask with tripsyn-EDTA but i didn't be able....
A last question, in your opinion can i use amphotericin B in my monocyte-macrophage culture?
well, i know the method to obtain PBMC with the Ficoll centrifugation, and to separate monocytes from other cells i allow PBMC to adhere to the bottom of flask with an overnight incubation at 37 °C and 5% CO2.
At the end, i have a lot of cells that are attached to the flask.
What i have to do is to differentiate them into macrophages and than detach the cells for the stimulation: how can i differentiate and detach them?
I've tryed to culture for about 6-7 days with RPMI 1640 + 20% of human serum, changing the medium every 2-3 days but i don't know if this have differentiated them...and than i've tryed to get them from the flask with tripsyn-EDTA but i didn't be able....
A last question, in your opinion can i use amphotericin B in my monocyte-macrophage culture?
See the first amaxa link here, it has differentiation media recipe on page 3 and nice illustrated protocol in its entirity.
http://search.vadlo.com/b/q?sn=158621799&a...phage&rel=0
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thank you for the precious reply... 