Methodology article

Study of membrane potential in T lymphocytes subpopulations using flow cytometry

Fernanda Mello de Queiroz^1,2 , Cristiano G Ponte^2,3 , Adriana Bonomo^4,5 , Rosane Vianna-Jorge^2,6 and Guilherme Suarez-Kurtz^2,7

¹  Molekulare Biologie Neuronaler Signale, Max-Planck-Institut für Experimentelle Medizin, Hermann-Rein-Strasse 3, 37075 Göttingen, Germany

²  Divisão de Farmacologia, Coordenação de Pesquisa, Instituto Nacional de Câncer, Rio de Janeiro, Brazil

³  Centro Federal de Educação Tecnológica de Química do Rio de Janeiro, Rio de Janeiro, Brazil

⁴  Divisão de Medicina Experimental, Coordenação de Pesquisa, Instituto Nacional de Câncer, Rio de Janeiro, Brazil

⁵  Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

⁶  Departamento de Farmacologia Básica e Clínica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

⁷  Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

author email corresponding author email

BMC Immunology 2008, 9:63doi:10.1186/1471-2172-9-63

Published:	3 November 2008

Abstract

Background

Ion channels are involved in the control of membrane potential (ψ) in a variety of cells. The maintenance of ψ in human T lymphocytes is essential for T-cell activation and was suggested to depend mostly on the voltage-gated Kv1.3 channel. Blockage of Kv1.3 inhibits cytokine production and lymphocyte proliferation in vitro and suppresses immune response in vivo. T lymphocytes are a heterogeneous cell population and the expression of Kv1.3 varies among cell subsets. Oxonol diBA-C4-(3) was used to determine ψ by flow cytometry. The presence of distinct T cell subsets was evaluated by immunophenotyping techniques and the contribution of Kv1.3 channels for the maintenance of ψ was investigated using selective blockers.

Results

The distribution of ψ in T lymphocytes varied among blood donors and did not always follow a unimodal pattern. T lymphocytes were divided into CD3⁺/CD45RO^- and CD3⁺/CD45RO⁺ subsets, whose peak channel values of ψ were -58 ± 3.6 mV and -37 ± 4.1 mV, respectively. MgTX (specific inhibitor of Kv1.3 channels) had no significant effect in the ψ of CD3⁺/CD45RO^- subsets but depolarized CD3⁺/CD45RO⁺ cells to -27 ± 5.1 mV.

Conclusion

Combination of optical methods for determination of ψ by flow cytometry with immuophenotyping techniques opens new possibilities for the study of ion channels in the biology of heterogeneous cell populations such as T lymphocyte subsets.