3 replies to this topic

[maninder]

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

[DZenker]

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.

[jadefalcon]

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

[thegradstudent]

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