Monocyte dissociation - Monocyte dissociation (Aug/25/2004 )
Hi. I am culturing monocytes obtained from fresh human blood and am having issues detaching them from the plasticware (pre coated with 4% albumin). I never seem to get all the cells off. Have tried EDTA, lignocaine and Gibco cell dissociation buffer. Can anyone tell me if it is normal to only recover a small amount of cells? Also is there a better way of dissociating them, i want to do FACS so can't use enzymes?
I am also facing similar problem. I am trying to isolate PMS by Ficoll gradient and then labeled it for CD14 and found that approx. 15% cells are monocytes. I am counting cells accordingly and putting it for experimentation. At the end of experiment I dissociate cells with 0.1X Trypsin EDTA. May be initially you should put 50000 cells so that you can get at least 20000 cells for flow purpose. Another way is to fix cells with 1% PFA for 37 deg for 1 hour and then scrap cells down and pellet it at 1000 rpm for 5 min. wash it and use it for flow.
I have tried these experiments with THP cell line and I am doing all standardization with MDMs.
Be in communication so we can share our experiences.