Isolation of rat megakaryocytes by immunomagnetic beads.
An immunomagnetic procedure was developed to purify rat megakaryocytes to homogeneity from flushed marrow cells. The cells from femurs, tibias, and humeri were initially centrifuged at low speed (800 rpm) to remove platelets and layered over a 1.050 g/cm3 Percoll density gradient. After washing at relatively high speed (1,500 rpm), rabbit anti-rat platelet serum (APS) was added to the cell suspension and incubated for 30 min at 4 degrees C. The cells were washed and subsequently treated with immunomagnetic beads coated with sheep anti-rabbit IgG antibody at room temperature for 10 min. Megakaryocytes were selectively isolated using a magnetic concentrator with a purity of 96.6 +/- 3.9%, recovery of 67.7 +/- 30.8%, and viability of 96.0 +/- 3.2%, although megakaryocytes accounted for 0.11 +/- 0.05% of starting marrow cells. Four to 7 x 10(6) megakaryocytes were obtained from 30 rats with a single population. This quantity provided us a possibility to characterize cytokine receptors. To determine if the nearly purified megakaryocytes were able to response to erythropoietin (Epo), one of purified promoting factors in megakaryocytopoiesis, the cells were incubated with 125I-labeled Epo at 15 degrees C for 90 min in the absence and presence of 100-fold excess unlabeled Epo. Autoradiographic analysis demonstrated the specific silver grains on the megakaryocytes, suggesting the presence of Epo receptors. Scatchard analysis revealed a single class of binding sites. These findings suggest that this method may be useful for megakaryocytic receptor studies of cytokines as well as the physiology or biochemistry of megakaryocytes.
Shikama
Y
Y
Department of Internal Medicine I, Fukushima Medical College, Japan.
D000818:Animals; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D007158:Immunologic Techniques; D008280:Magnetics; D008297:Male; D008533:Megakaryocytes / Q000166:cytology*; D008863:Microspheres; D051381:Rats; D011919:Rats, Inbred Strains