4 replies to this topic

[hollegaard]

Hi,

I have a little problem with understanding the selection procedure of monocytes from PMBC decriebed at this forum.

I understand the selection procedure, using Leucoprep tubes with Neucosep 1.077, but my problem is the next step where it is suggested to let the cells incubate for a few hours and then wash away the media, since the monocytes should adhere to the plastic surface???
I have been working with THP-1 cells for a while now and every time I have seen adherent cells it is because that they are differentiating. If they differantiate to macrophages they arent much worth as monocytes I guess... Could anyone here tell me if I am wrong?

My general problem is that I, am going to isolate monocytes from whole blood, so if anyone knows how to do this without differntiating the cells, I would very gratefull to know.  Ill have to be able to culture the monocytes afterwards..

Thanks,
Mads

[fred_33]

hi
maybe can you try this one :

http://www.protocol-...ive/7/1891.html

[hollegaard]

Well, thats the discussion that I have a problem with, since they (besides the magnetic bead part) suggests that you select the monocytes by washing away the nonadherent cells after purification with luecoprep.
Thats the reason why I called my question "isolating monozytes from wholeblood 2". because I dont understand how you select the monocytes out of the culture if they differntiate first. I mean if they differentiate to adherent cells, they must be macrophages and I need pure monocytes to my experiments.

[sakrob]

hollegaard, on Feb 28 2005, 01:57 AM, said:

I have been working with THP-1 cells for a while now and every time I have seen adherent cells it is because that they are differentiating. If they differantiate to macrophages they arent much worth as monocytes I guess... Could anyone here tell me if I am wrong?

My general problem is that I, am going to isolate monocytes from whole blood, so if anyone knows how to do this without differntiating the cells, I would very gratefull to know.  Ill have to be able to culture the monocytes afterwards..

<{POST_SNAPBACK}>

what r u using to differentiate the the cell and wt is the concentration>

[kberrada]

I just saw your question pertaining to monocyte isolation. You ar right.  Macrophages will adhere to dishes and that is a very quick way to get rid of macrophages if you don'y need them for your experiment.

A better way to isolate monocytes from whole blod is to isolate PBMCs using percoll gradient, followed by elimination of macrophages.  Then you capture monocytes using specific beads from R&D.  These beads are suppose to capture monocytes by binding to specific monocyte markers.

Good luck