A mixing chamber to enucleate avian and fish erythrocytes: preparation of their plasma membrane

Biochemical studies of the plasma membrane and the cytoskeleton of nucleated erythrocytes are strongly limited by the difficulties encountered in enucleating large quantities of cells. We describe an easily built hydrodynamic system which allows rapid preparation of large amounts of avian and fish erythrocyte plasma membranes. The contents of two 25-ml syringes containing hemolyzed nucleated erythrocytes are forced through four capillaries to a specially designed mixing chamber which fills a collecting syringe. The 50-ml erythrocyte suspension can be processed in 2 s. The high speed flow is achieved with a hand-activated piston. The turbulences in the mixing chamber are carried to an optimal efficiency by the vis-à-vis disposition of the four mixing jets. The enucleated membranes are separated from the nuclei and residual nucleated cells by differential centrifugations. They do not show contamination with nuclear material. Erythrocytes from chicken and trout have been used. They present striking differences in their stability toward hydrodynamic disruption, erythrocytes from chicken being far more stable. Ninety-five percent of trout erythrocytes are enucleated after only one run through the mixing chamber. Two runs performed at the maximal flow rate are necessary to enucleate chicken erythrocytes with a yield of 80%. In the former case most of the purified enucleated plasma membranes are fragmented in small vesicles while they retain a large size in the case of chicken erythrocytes. The proteins of the membranes thus prepared are characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: we found that erythrocyte membranes from trout are remarkable for their small spectrin content compared to those from chicken.