Measurement of Splenic T Cell Membrane Potential

Isolated splenic CD4⁺ T cells were cultivated in an RPMI 1640 medium that was supplemented with 10% heat-inactivated fetal calf serum (Merck, Darmstadt, Germany), antibiotics (penicillin and streptomycin mixture, Fujifilm Wako Pure Chemicals), Con-A (5 µg/mL), and IL-2 (10 U/mL) for 48 h. Membrane potentials were measured by using the voltage-sensitive dye DiBAC₄(3), as previously reported [9 (link),38 (link)]. Changes induced in the fluorescent intensity of DiBAC₄(3) by alkaline pH (pH 8.5) were measured with an ORCA-Flash2.8 digital camera (Hamamatsu Photonics, Hamamatsu, Japan). Data collection and analyses were performed by using an HCImage system (Hamamatsu Photonics). Images were obtained every 5 s.

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Endo K., Kito H., Tanaka R., Kajikuri J., Tanaka S., Elboray E.E., Suzuki T, & Ohya S. (2019). Possible Contribution of Inflammation-Associated Hypoxia to Increased K2P5.1 K+ Channel Expression in CD4+ T Cells of the Mouse Model for Inflammatory Bowel Disease. International Journal of Molecular Sciences, 21(1), 38.

Publication 2019

heat-inactivated fetal calf serum penicillin and streptomycin mixture ORCA-Flash2.8 digital camera HCImage system

Antibiotics Camera Cd4 t cells Con a Dibac4 Fetal calf serum Membrane potentials Orca Penicillin Splenic Streptomycin

Corresponding Organization : Nagoya City University

Other organizations : Kyoto Pharmaceutical University, Osaka University, South Valley University

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Variable analysis

independent variables

Cultivation of isolated splenic CD4+ T cells in RPMI 1640 medium
Supplementation of the medium with 10% heat-inactivated fetal calf serum
Supplementation of the medium with antibiotics (penicillin and streptomycin mixture)
Supplementation of the medium with Con-A (5 µg/mL)
Supplementation of the medium with IL-2 (10 U/mL)
Cultivation time of 48 h

dependent variables

Changes in membrane potentials of the CD4+ T cells, as measured by the voltage-sensitive dye DiBAC4(3)
Changes in the fluorescent intensity of DiBAC4(3) induced by alkaline pH (pH 8.5)

control variables

Use of the voltage-sensitive dye DiBAC4(3) to measure membrane potentials, as previously reported [9, 38]
Use of an ORCA-Flash2.8 digital camera (Hamamatsu Photonics) to measure changes in fluorescent intensity
Use of an HCImage system (Hamamatsu Photonics) for data collection and analysis

positive controls

None mentioned

negative controls

None mentioned

Annotations

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