The Dynal Negative Monocyte Isolation Kit is a way of isolating resting monocytes from a mononuclear cell (MNC) preparation. I have used the Dynal kit extensively within our lab during my graduate work. The principle of the monocyte isolation kit is the use of uniform superparamagnetic polystyrene beads (Dynabeads). The Dynabeads are coated with an Fc specific human antibody against mouse IgG. This, in conjugation with the supplied antibody mix (which contains a mixture of mouse IgG monoclonal antibodies for human CD2, CD7, CD16, CD19, CD56 and CD235a) enables the extraction of contaminating, non-monocytic cells. In addition, a gamma globulin blocking reagent is able to block Fc receptors on monocytes, preventing any binding of monocytes to Dynabeads. Therefore with the ‘magnetic particle concentrator’ (which has to be purchased separately), all cells bound to the magnetic Dynabeads are forced to one side of a tube, enabling the easy removal of negatively isolated monocyte populations.
The procedure for isolating monocytes from whole blood is relatively easy and takes around 45 minutes after MNC preparation. The instructions for use accompanying the kit are reasonably self explanatory; however there was a lack of information concerning the best method for MNC isolation. Within our lab we isolated MNC using a standardized technique with Lymphoprep (Axis-Shield), layered with fresh whole blood.
The manufacturers state that the average monocyte purity is around 80%, however surprisingly after routinely isolating monocytes daily we achieved greater than 90% purity. We also analyzed surface CD11b expression by flow cytometry throughout the isolation procedure, in order to examine any artefactual activation due to the Dynabead incubation or experimental procedure. We found there was no significant increase in monocytic CD11b expression throughout the method and were able to achieve resting populations of isolated monocytes. In order to achieve greater purity and increased yields of monocytes, we found coating all plasticware including pipette tips with SigmaCote (Sigma) and incubating with the blocking reagent for around 1 minute helped, which was not stated within the instructions. We also found the use of the MPC-S magnetic particle concentrator cumbersome, as with greater numbers of MNC it was difficult to keep washing 5 eppindorfs continuously. Therefore to increase yields we found the larger MPC-L using 15ml tubes was more efficient and far easier to handle as only one tube would be in use.
In summary the monocyte negative isolation kit from Dynal, is effective at isolating relatively high yields of resting pure monocyte populations from whole blood. The only downside is the price of the product, which can prove costly when large amounts of differing blood samples is needed. However the procedure can guarantee good populations of monocytes, which, at times, is priceless.
KJ Woollard, Ph.D.
Senior Research Officer
Baker Medical Research Institute
Australia