hi, Can anyone suggest a method to isolate monocytes from whole blood?

Yeah, its quite easy with the ficoll-hypaque method. Dilute your buffy coat 1:2 with PBS. Prepare some 50 ml tubes with 25 ml ficoll (density 1.077 g/ml), then overlay the ficoll with an equal amount of the mixture from above. Centrifuge for 30 min with 700 x g without brake at 4 degress C. Soak up the white interphase between the plasma fraction and the ficoll fraction with a pipette and transfer it into a fresh tube. Wash twice with PBS. Then resuspend the cell pellet in medium and transfer the whole stuff to cell culture dishes (about 5 million PBMC per ml). The resulting PBMC suspension contains about 10% monocytes and 90% lymphocytes. Let the monocytes adhere for 1 h and wash away the non-adherent lymphocytes, so you get a nearly pure monocyte culture.

There's a commercial variation of the Ficoll Isopaque from Greiner we use for the Preparation of PBMC's from whole blood.

That System is called LeucoSep ?.

It includes 50ml Falcon Tubes containing a filter disk, so the seperation of MNC's and Ery's is near perfect.

Should be available at www.greiner-lab.com

Mike

You can also purify them through magnetic beads, stsrting from PBMC isolated as previously said. I recomend CD14 Microbeads from Miltenyi Biotec.

hi,
this paper gives nice idea about the technique " isolation of pure monocytes"
http://memorias.ioc.fiocruz.br/952/3879.html

hope it helps you.
gud luck

Hello,

Can I also use this method to purify blood cells from bone marrow specimen? Thank you.

Freshman using Ficoll-Hypaque

Dear payeli,

Thanks for your sharing,
I'm new to the isolation of monocytes.

From the article you recommended,
I've learned that the improved procedure comprises 2 major steps, one is Ficoll-Hypaque and the other is iso-osmotic Percoll gradient.

I'm much clear with the Ficoll-Hypaque procedure but not with the second step.

Can you or someone please advise the detailed procedure after removing the monocyte and lymphocyte layer from the centrifuged Ficoll-Hypaque gradient?

e.g. what's the volumn ratio when mixing the PBMC isolate into iso-osmotic Percoll solution?
After centrifugation, will there be several layers in the Percoll gradient?
Where will be the monocytes enriched layer that I should retreive?

Please provide some suggestions, thanks a lot!!

BR

Tim


[quote name='payeli' date='Mar 13 2006, 08:09 PM' post='43996']
hi,
this paper gives nice idea about the technique " isolation of pure monocytes"
http://memorias.ioc.fiocruz.br/952/3879.html

hope it helps you.
gud luck