A new method for the preperative and analytical electrophoresis of cells

Abstract

Abstract:In this paper, a new method is described for the horizontal electrophoresis of cells on a density cushion under near-isopycnic conditions. When cell sedimentation is minimized, the electrophoresis of red blood cells (RBC) used as model cells within an anti-convective porous matrix (with pores over 300 μm in diameter) was capable of separating a mixture of human and chicken RBC according to their electrophoretic mobilities. Samples taken from the separated RBC bands show over 90% purity for each species. The simultaneous electrophoresis of several RBC samples carried out under identical conditions permitted the use of comparative data based on the electrophoretic mobility of cells which differ in their surface properties. We believe that this relatively simple system, in which cell sedimentation and convection are minimized, has the potential to be modified and adapted for the separation of other cell types/organelles.

Cell separation with horizontal cell electrophoresis under near-isopycnic conditions on a “density cushion”

“…The preparation of human and chicken RBC was described in an earlier paper [12]. The human eosinophilic leukaemia cell line Eol-1 (DSMZ ACC386) was cultured in RPMI-1640 medium (Sigma, St Louis, MO/USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco Lab., NY/USA), 10 μg/ml streptomycin (Polfa Tarchomin, Poland), 100 IU/ml penicillin (Polfa Tarchomin, Poland), and 10 μg/ml neomycin (Polfa Tarchomin, Poland).…”

“…The first system was designed for the separation of RBC in a stabilizing PU sponge; the apparatus and the preparative procedures were previously described [12]. The new apparatus was based on the previous system, with the main improvements being the depth of the separation chamber (10 mm instead of 4 mm) and the composition of the electrophoresis solutions being designed to provide a biocompatible "density cushion".…”

“…The compositions of the solutions used for system I with the PU sponge were described earlier [12]. For the electrophoretic separation of cells on the "density cushion" (system II), the solutions were modified, but their compositions were also based on dextran MW 15000-20000 (MP Biomedicals LLC, Eschwege, Germany), sucrose (PoCh, Gliwice, Poland) and PBS (Biomed, Lublin, Poland); the reasons for these choices were also previously described [12].…”

“…Preparative cell electrophoresis was achieved with the free-flow curtain apparatus [10], which is now commercially available [11]. Our earlier paper described a horizontal electrophoresis system which effectively separated human and chicken red blood cells (RBC), which differ by about 30% in their electrophoretic mobilities [5,12]. The separation was carried out under near-isopycnic conditions, when cell sedimentation is negligible, within an anticonvective porous matrix of polyurethane sponge with a pore size over 300 μm.…”

“…The separation was carried out under near-isopycnic conditions, when cell sedimentation is negligible, within an anticonvective porous matrix of polyurethane sponge with a pore size over 300 μm. This method permitted a comparative electrophoretic study of several samples in parallel [12]. The polyurethane (PU) matrix was found to stabilize the electrophoretic system (system I), and is suitable for studies with red blood cells and lymphocytes.…”