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Here, we present a protocol for a novel gap junction intercellular communication assay designed for the high-throughput screening of gap junction-modulating chemicals for drug discovery and toxicological assessment.
Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and pathological roles, there is need of high-throughput screening (HTS) assays to identify GJ modulators in drug discovery and toxicology assays. A novel iodide-yellow fluorescent protein-gap junction-intercellular communication (I-YFP-GJIC) assay fulfills this need. It is a cell-based assay including acceptor and donor cells that are engineered to stably express a yellow fluorescent protein (YFP) variant, whose fluorescence is sensitively quenched by iodide, or SLC26A4, an iodide transporter, respectively. When iodide is added to a mixed culture of the two cell types, they enter the donor cells via the SLC26A4 transporter and diffuse to the adjacent acceptor cells via GJs where they quench the YFP fluorescence. YFP fluorescence is measured well by well in a kinetic mode. The YFP quenching rate reflects GJ activity. The assay is reliable and rapid enough to be used for HTS. The protocol for the I-YFP-GJIC assay using the LN215 cells, human glioma cells, is described.
Gap junctions (GJs) act as intercellular channels to allow the diffusion of small molecules of <1 kDa such as nutrients, metabolites, and signaling molecules between adjacent cells. The junctional elements include a hemichannel or connexon in each cell, and each connexon constitutes six connexins (Cxs)1. GJs and Cxs have been used in toxicology assays of carcinogens such as polycyclic aromatic hydrocarbons (PAH), which are GJ inhibitors2,3,4. Disrupted GJIC has been associated with nongenotoxic carcinogenesis5,
1. Generation of lentiviruses expressing YFPQL and SLC26A4
Twenty-nine 96-well culture plates were used to screen 2,320 chemicals to identify novel GJIC modulators by I-YFP GJIC assay using the LN215-YFPQL and LN215-I− cells. The results obtained with a representative plate are shown in Figure 2. The percentage of YFP fluorescence in each well is shown as a line graph in Figure 2A and the percentage of GJIC activity in each well is shown in th.......
The I-YFP-GJIC assay can be used for HTS because it is robust, rapid, and inexpensive. An HTS assay is considered robust if the Z'-factor is above 0.525. See Zhang et al. for a description of the statistical analysis used to assessing the suitability of HTS assays25. When LN215 cells were used, the Z'-factor was >0.5 without any assay optimization. If other cell types are used in the assay and its Z'-factor is <0.5, the robustness can be improved by exte.......
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2011-0023701, 2016R1D1A1A02937397, and 2018R1A6A1A03023718).
....Name | Company | Catalog Number | Comments |
96-well plate | SPL | 30096 | |
Calcium chloride (CaCl2) | Sigma | C5670 | I-solution |
D-(+)-Glucose | Sigma | G7021 | C-solution, I-solution |
Dimethyl sulfoxide (DMSO) | sigma | 276855 | |
HEPES | Sigma | RES6003H-B7 | C-solution, I-solution |
Lipofectamine 2000 | Invitrogen | 11668-027 | transfection reagent |
Magnesium chloride hexahydrate (MgCl2 6H2O) | Sigma | M2393 | C-solution |
Microplate reader | BMG LabTech | POLARstar Omega 415-1618 | |
pMD2.GÂ | Addgene | #12259 | |
Polybrene | sigma | H9268 | |
Poly-L-lysine solution | sigma | P4707 | |
Potassium chloride (KCl) | Sigma | P5405 | C-solution, I-solution |
psPAX2Â | Addgene | Â #12260 | |
Puromycin Dihydrochloride | sigma | P8833 | |
Sodium chloride (NaCl) | Sigma | S5886 | C-solution, I-solution |
Sodium hyroxide (NaOH) | Sigma | S2770 | |
Sodium Iodide (NaI) | Sigma | 383112 | I-solution |
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