Single-step enrichment of basophils from human peripheral blood by a novel method using a Percoll density gradient.
We have developed a novel continuous flow-through cell separation method using a Percoll density gradient. This method can continuously separate a large number of cells into five fractions according to their densities. To apply this method to the separation of basophils, Percoll density gradients were modified to improve basophil enrichment. When a set of Percoll density gradients was prepared (1.071, 1.075, 1.080, 1.084, and 1.090 g/mL) the basophils in a healthy volunteer were enriched by an average of 23.1 and 63.5% at Percoll densities of 1.075 (fraction 3) and 1.080 g/mL (fraction 4), respectively. On average, the yield of basophils was 1.66 × 10(5) cells in fraction 3 and 1.61 × 10(5) cells in fraction 4 from 9 mL of peripheral blood. The expression of CD203c (cluster of differentiation 203c) on separated basophils was upregulated by anti-immunoglobulin E stimulation similar to basophils in whole blood. Histamine release induced by calcium ionophore was also observed in the separated basophils. The present method will be useful for basophil enrichment since it preserves their function without using counterflow elutriation and immunological reagents, and this method will be effective as a preparative separation for cell purification by flow cytometry.
Department of Physiology, Aichi Medical University School of Medicine, Yazako, Nagakute-city, Aichi, Japan.
Hiroyuki
H
Shiono
Department of Physiology, Aichi Medical University School of Medicine, Yazako, Nagakute-city, Aichi, Japan.
Takuya
T
Matsui
Department of Physiology, Aichi Medical University School of Medicine, Yazako, Nagakute-city, Aichi, Japan.
Tadashi
T
Okada
Bioseparation Technology Laboratory, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
Yoichiro
Y
Ito
D001491:Basophils / Q000737:chemistry*; D002469:Cell Separation; D002499:Centrifugation, Density Gradient / Q000295:instrumentation; D064368:Healthy Volunteers; D006801:Humans; D007958:Leukocyte Count; D011205:Povidone / Q000737:chemistry*; D012822:Silicon Dioxide / Q000737:chemistry*
C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone
Basophils; Cell separation; Centrifugation; Density gradient; Human peripheral blood