Immunomagnetic separation is a suitable method for electrophysiology and ion channel pharmacology studies on T cells

Background:Ion channels play emerging role in the physiological and pathological function in many cell types, including the non-excitable immune cells. As immune cells represent a heterogeneous population with considerable functional diversity, subtype-specific functional studies, such as single-cell electrophysiology of ion channels (patch-clamp), require proper subset identification and separation. Magnetic-activated cell sorting (MACS) techniques provide an alternative to fluorescence-activated cell sorting (FACS), however, the potential impact of MACS on patch-clamp experiments were not studied yet. Among others, the presence of MACS-related beads on the cell surface may alter the biophysical and pharmacological parameters of the ion channels expressed in the membrane. This motivated our experiments to describe the feasibility of MACS for electrophysiology studies on immune cells. We examined the biophysical and pharmacological parameters of the voltage gated Kv1.3 K+ channel in activated CD4+ T-cells as well as the membrane capacitance following immunomagnetic positive separation, using the REAlease® kit. This kit allows three experimental configurations: bead-bound configuration, bead-free configuration following the removal of magnetic beads and the label-free configuration following removal of the REAlease® complex congaing the antibody fragment that recognizes CD4. As controls we used FACS separation as well as immunomagnetic negative selection.Results:We found similar purity and cell viability using FACS or MACS-based positive (REAlease®) and negative selection approaches. The membrane capacitance and of the biophysical parameters of Kv1.3 gating, voltage-dependence of steady-state activation and inactivation kinetics of the current were oblivious to the presence of the beads or any components of the REAlease® kit on the cell surface. We found subtle differences in the activation kinetics of the Kv1.3 current in cells obtained using various cell isolation configurations that could not be explained by the presence of MACS-related compounds on the cells. Neither the equilibrium block of Kv1.3 by TEA or charybdotoxin (ChTx) nor the kinetics of ChTx block are affected by the presence of the magnetics beads on the cell surface. Conclusions:Taken together our results support, that MACS is a suitable method for studying ion channel in non-excitable cells, such as T-lymphocytes with the benefit of easy application and relatively low instrumentation costs.