Immunomagnetic cell separation is a suitable method for T cell electrophysiology and ion channel pharmacology

Abstract

Abstract Immunomagnetic cell separation is a powerful tool of immunology, however the presence of beads on the cell surface might alter the electrophysiological and pharmacological parameters of the ion channels expressed in the separated cells. We addressed this issue by measuring the aforementioned properties of Kv1.3, the predominant voltage-gated K+ channel of activated T cells upon immunomagnetic separation. PBMCs from healthy donors were activated then CD4+ cells were separated with REAlease® CD4 Kit (Miltenyi Biotec Inc.). As control, we made stepwise removal of REAlease® antibody-bead complex, immunomagnetic negative separation and FACS. Purity and viability was determined by flow cytometry. Electrophysiology studies were made with patch-clamp technique in whole-cell configuration. The presence of bead-antibody complex statistically significantly increased the time constant (τact) characterizing the activation kinetics of the current as compared to negative selection (p=0.049) and antibody only (p=0.024) controls, however the increase in τact from ~0.5 ms to ~0.6 ms does not bear any biological significance. Other gating parameters of Kv1.3, such as inactivation time constant and midpoint of voltage-dependence of steady-state activation were unaffected by the beads. Moreover, the binding of a peptide (ChTx) and a small organic molecule (TEA) inhibitor to Kv1.3 was oblivious to the presence of beads. In addition, the electrical capacitance of the cell membrane was not altered when beads were attached to the cells. Based on our results we conclude that the application of immunomagnetic cell separation is completely suitable to electrophysiology studies of immune cells.