Improved isolation of normal human reticulocytes via exploitation of chloride-dependent potassium transport.
Studies on normal human reticulocytes have been limited by a lack of methods for effective reticulocyte enrichment. This study shows a convenient new approach for selective enrichment of reticulocytes from normal blood samples. We have developed a modified arabinogalactan density gradient that contains high potassium levels, approximating the internal cation composition of red blood cells (RBC). The low-density populations from this gradient are enriched in reticulocytes, and the highly selected lowest density fraction shows a much higher reticulocyte enrichment than that obtained with high sodium chloride arabinogalactan density gradients, or other previously reported density gradient methods. We found that this improved isolation is caused by suppression of potassium loss and reticulocyte dehydration via chloride (KCI) cotransport. When the low-density fraction of RBC from a high-potassium gradient was subsequently incubated in high sodium chloride medium and reseparated on a sodium chloride density gradient, the reticulocytes dehydrated and were recovered in high-density fractions. The highest-density fractions from this secondary gradient yield 95% to 99% reticulocytes. We anticipate that this method will benefit investigators who require reticulocyte enriched populations for a wide variety of applications.
Sorette
M P
MP
Department of Laboratory Medicine, University of California, San Francisco 94143.
Shiffer
K
K
Clark
M R
MR
D001692:Biological Transport; D002469:Cell Separation / Q000379:methods; D016922:Cellular Senescence; D002499:Centrifugation, Density Gradient; D002712:Chlorides / Q000502:physiology; D003598:Cytoskeletal Proteins; D004912:Erythrocytes / Q000166:cytology; D006441:Hemoglobin A / Q000032:analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000032:analysis; D009479:Neuropeptides; D011188:Potassium / Q000378:metabolism; D011990:Receptors, Transferrin / Q000032:analysis; D012156:Reticulocytes / Q000166:cytology*; D014882:Water-Electrolyte Balance
D002712:Chlorides; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D009479:Neuropeptides; D011990:Receptors, Transferrin; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; D006441:Hemoglobin A; D011188:Potassium