Study of membrane potential in T lymphocytes subpopulations using flow cytometry

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.

Membrane potential of CD4+ T cells is a subset specific feature that depends on the direct cell-to-cell contacts with monocytes

“…Statistical details are presented in main text. ory CD3 ϩ T cells exhibited different patterns of PMP and responded with various grades of depolarization to MgTX [16]. In addition, the report by Mello de Queiroz suggested the existence of two distinct subsets within the pool of memory lymphocytes with different PMP and responsiveness.…”

The functional contribution of calcium ion flux heterogeneity in T cells

“…Furthermore, a subsequent paper analysed Ca 2+ oscillations in different cell regions and found that knocking down type 3 RyRs decreased oscillatory frequency in the cytosol whereas chemical inhibition of RyRs reduced oscillation in all regions of the cell including the cell border, cytosol and nucleus 18 . However, the focus on store depletion is incomplete without addressing the mechanisms of store replenishment via sacro‐endoplasmic reticulum Ca 2+ ‐ATPase (SERCA) channels found on the ER membrane 21 . Evidently, the extent of ER refilling by SERCA channels is inversely proportional to the global Ca 2+ elevation 12 .…”

“…However, a critical element to maintain the calcium influx is the electrochemical driving force produced by potassium (K + ) and the most commonly reported types are voltage‐gated (K v ) and Ca 2+ ‐activated (K Ca ) channels. In a resting state, the membrane potential of the cell is maintained at ~65 mV by the efflux of K + via K v channels, and Ca 2+ transport into the ER 21 . The influx of Ca 2+ via CRAC channels, which are considered as inward rectifiers 3 due to their ability to more readily pass Ca 2+ into the cell, can generate a depolarising Ca 2+ current.…”

Kinetic Measurements Using Flow Cytometry: New Methods for Monitoring Intracellular Processes

“…The cells were loaded with 300 nM di-BA-C4-(5) for 8 min at 37°C. 3 Positive control at the end of the measurements is not necessary as this dye is not compartmentalized and not extruded by the glycoprotein efflux pump either 3 (for details, see Table 1). Compensation was 6.56% for Fluo-3-AM-di-BA-C4-(5) and 17.69% for di-BA-C4-(5)-Fluo-3-AM.…”