An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay

Summary

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.

Abstract

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.

Introduction

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 inhibitors^2,3,4. Disrupted GJIC has been associated with nongenotoxic carcinogenesis^5,6. As a potential therapeutic target, GJ involvement has been reported in particular subtypes of seizures^7,8, protection from cardiac and brain ischemia/reperfusion injury⁹, migraine with aura¹⁰, drug-induced liver injury^6,11, and wound healing¹². High-throughput screening (HTS) assays are required to identify GJ-modulating chemicals or antibodies for drug discovery, for toxicology assays, and to identify novel cellular regulators of GJ activity. HTS assays can also be used to investigate structure-activity relationships of GJ modulators^2,13,14,15.

Some GJIC assays include dye transfer or dual patch clamp techniques. Lucifer yellow CH (LY) and calcein acetoxymethyl ester (calcein-AM) have been used in dye-transfer assays. Cells are not permeable to LY, which is introduced by microinjection, scrape loading, or electroporation. Once inside the cell, LY spreads into neighboring cells via GJs and GJ activity is assayed by the extent of the LY migration¹⁶. Calcein-AM assays usually involve gap-fluorescence recovery after photobleaching^17,18. Calcein-AM is a cell-permeant dye that is converted intracellularly into impermeable calcein by an intrinsic esterase. The assay requires a confocal microscope to observe the transfer of calcein-AM into a cell from those surrounding it following laser photobleaching. If functional GJs are present, calcein-AM in adjacent cells enters the photobleached cells and the fluorescence is recovered. GJ activity is assayed by the extent of fluorescence recovery of the photobleached cells. Dye-transfer assays are laborious and time consuming or have low sensitivities. Dual patch clamping is an electrophysiological method that measures junctional conductance. It is relatively sensitive, with a direct dependence of conductance on the number of open GJs¹⁹; however, it is technically demanding, time consuming, and expensive²⁰. The I-YFP-GJIC assay was developed for use in HTS.

Figure 1 illustrates the components and steps of the I-YFP GJIC assay, which utilizes acceptor cells expressing an iodide-sensitive YFP variant bearing H148Q and I152L (YFP^QL) and donor cells expressing an iodide transporter (SLC26A4)²¹. The two mutations carried by YFP^QL allow quenching of fluorescence by iodide²². Iodides are added to co-cultured acceptor and donor cells; they do not enter the acceptor cells, but are taken up by the SLC26A4 transporters present on the donor cells. Iodides in the donor cells diffuse through functioning GJs into adjacent acceptor cells where they quench the YFP^QL fluorescence. If GJs are closed or blocked by inhibitors, iodide cannot enter the acceptor cells to quench the fluorescence. The YFP^QL quenching rate reflects GJ activity. The I-YFP GJIC assay procedure is neither complicated nor time consuming. It is compatible with HTS and can be used to test the effects of a large number of compounds on GJ activity in a relatively short period. It requires only acceptor and donor cells, and two balanced salt solutions. The protocol described below is based on LN215 cells whose major Cx is Cx43²¹. The LN215-YFP^QL receptor and LN215-I⁻ donor cells were generated by transduction with lentiviruses expressing YFP^QL or SLC26A4^21,23.

Protocol

1. Generation of lentiviruses expressing YFP^QL and SLC26A4

1. Grow HEK293T human embryonic kidney cells to 80% confluency on 100 mm culture plates. Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin is the culture medium used throughout the protocol to maintain HEK293T and other cells mentioned below.
2. Coat 6-well culture plates by adding 2 mL of 0.005% of sterile poly-L-lysine (PLL) solution to each well for 10 min. Aspirate the PLL solution and rinse the surface twice with 2 mL of sterile water.
3. Wash the HEK293T cells with 10 mL of phosphate buffered saline (PBS) and treat the cell monolayers in each 100 mm dish with 2 mL of 0.25% trypsin-EDTA solution at 37 °C for 3 min. Add 5 mL of culture medium and resuspend the cells.
4. Count the cells in a hemocytometer and adjust the density of the cell suspension to 250,000 cells/mL in culture medium and add 500,000 cells in 2 mL of culture medium to each lysine-coated well of 6-well plates. Incubate the cells in a humidified 5% CO₂/95% air atmosphere at 37 °C for 24 h and then replace the culture medium with DMEM without penicillin or streptomycin.
5. In a 1.5 mL tube, dilute 20 µL of transfection reagent with 500 µL of DMEM without serum or antibiotics. Mix gently by pipetting and let stand at room temperature for 5 min.
6. Meanwhile, pipette 250 µL of DMEM into each of two 1.5 mL tubes and then add 1500 ng of pLVX-EIP-YFP^QL or pLenti6P-SLC26A4, 1225 ng of psPAX2 and 375 ng of pMD2.G to each. The two lentiviral plasmids have been previously described²¹. Add 250 µL of diluted transfection reagent to each plasmid tube, mix gently and incubate for 20 min at room temperature.
7. After 20 min, add 500 µL of transfection reagent and plasmid complexes in the 1.5 mL tubes dropwise to each culture plate well in step 1.4 and mix by rocking the plate back and forth. Incubate cells at 37 °C in a CO₂ incubator for 12 h.
8. Replace the medium with 2.5 mL of fresh medium and incubate for an additional 48 h. Then place the culture plate on ice for 5 min to keep the conditioned medium containing lentivirus chilled to maintain infectivity.
9. Harvest the media containing lentiviruses and transfer to 15 mL conical tubes. Centrifuge at 3,000 x g at 4 °C for 3 min and then remove floating HEK293T cells from the supernatant by filtration at 0.4 µm.
10. Store the media containing lentiviruses at 4 °C for use within 2 days. For later use, store 200 µL aliquots at −80 °C.

2. Generation of LN215-YFP^QL and LN215-I⁻ cells by lentiviral transduction

1. Grow LN215 cells in 100 mm culture plates to 80% confluency in DMEM supplemented with 10% fetal bovine serum (FBS) 100 U/mL penicillin, and 100 µg/mL streptomycin as described above.
   NOTE: If the I-YFP-GJIC assay is conducted using a different cell line, use the appropriate culture medium. LN215-YFP^QL, and LN215-I^- cells can be provided by the University-Industry foundation, Yonsei University. Please contact the corresponding author.
2. One day before transduction, wash the cells twice with 10 mL of PBS, treat with 2 mL of 0.25% trypsin-EDTA at 37 °C for 3 min. Resuspend the cells in 5 mL of culture medium with a 10 mL serologic pipette and adjust the density to 50,000 cells/mL. Add 20,000 cells in 400 µL of media to each well of a 24-well culture plate for treatment as no virus control, YFP^QL, and SLC26A4 cells.
3. After 24 h of incubation at 37 °C, transduce two wells by replacing the culture medium with 400 µL of a 1:1 mixture of pLVX-EIP-YFP^QL or pLenti6P-SLC26A4 lentivirus and fresh culture medium supplemented with polybrene at a final concentration of 4 µg/mL. For no virus controls, replace with culture medium.
4. Incubate the cells at 37 °C for 15 h, aspirate the medium containing lentiviruses, add fresh culture medium, and incubate the cells for an additional 72 h.
   CAUTION: To prevent contamination of lentivirus between wells, use new tips or pipets for each well when you aspirate culture medium containing lentivirus or dispense fresh growth medium.
5. Wash the cells in each well twice with 0.5 mL of PBS, treat with 300 µL of trypsin-EDTA for 3 min. Resuspend the cells in 2 mL of culture medium and plate in six-well culture plates with 2 µg/mL puromycin.
6. Culture the cells in media containing 2 µg/mL puromycin until all cells in the control well are dead (round-shaped or floating when observed in microscope), which usually takes a week. Refresh the culture media containing puromycin every other day during the selection period. If LN215-YFP^QL or LN215-I^- cultures become confluent before selection has completed, transfer the cells to 100 mm plate and continue selection as in step 2.5.

3. Preparation of solutions required for the assay

1. Prepare 500 mL of C-solution (10 mM HEPES, 140 mM NaCl, 10 mM glucose, 5 mM KCl, 1 mM MgCl₂, and 1 mM CaCl₂) and 500 mL of I-solution (10 mM HEPES, 140 mM NaI, 10 mM glucose, 5 mM KCl, and 1 mM CaCl₂).
2. Adjust the pH of both solutions to 7.4 with 1 N NaOH, sterilize the solutions by filtrations at 0.4 µm for storage. Store at 4 °C for up to 1 month. Check the pH before using.

4. Plating the LN215-YFP^QL and LN215-I⁻ cells

1. Culture LN215-YFP^QL and LN215-I⁻ cells in 100 mm plates separately in culture medium to reach the populations required for assay. LN215-YFP^QL and LN215-I⁻ cells in 40% and 80% confluency in 100 mm plates, respectively, are sufficient for a 96-well plate assay.
2. One day before conducting the I-YFP GJIC assay, wash each 100 mm culture plate with 10 mL of PBS. Treat each plate with 2 mL of 0.25% trypsin-EDTA solution and incubate at 37 °C for 5 min. Resuspend the cells in each plate in 4 mL of culture medium and transfer to 15 mL conical tubes.
3. Pellet the cells by centrifugation at 1,000 x g for 3 min. Discard the supernatant and resuspend each cell pellet with 5 mL of culture medium. Break up any cell clumps into single cells by pipetting up and down about 20 times with a 10 mL serological pipette.
4. Count the cells in a hemocytometer, and dilute the cells in the culture medium to make cell suspensions of LN215-YFP^QL at 80,000 cells/mL and LN215-I⁻ at 160,000 cells/mL.
5. Mix 7 mL of LN215-YFP^QL and 7 mL of LN215-I⁻ cell suspensions in a reservoir. Add 100 µL of the mixture to each well of a 96-well cell culture plate using a multichannel pipette.
   NOTE: To add 100 µL of the mixture in each well of 96-well cell plate, about 10 mL of mixed cell suspension is needed. It is recommended to make more cell suspension than needed.
6. Incubate the cells in humidified 5% CO₂/95% air at 37 °C for 24 h. The LN215-YFP^QL and LN215-I⁻cell culture should be 100% confluent when the assay is conducted.

5. Conducting the I-YFP GJIC assay

NOTE: Use a fluorescence microscope with 20x magnification, and a GFP filter to check the 96-well plates to be sure there are no clumps of LN215-YFP^QL or LN215-SLC26A4 cells and that the cell cultures are fully confluent and well distributed before conducting the assay.

1. At least 30 min before doing the assay, turn on a microplate and set to 37 °C.
2. Wash the tubing of an automated injector with 3 mL of 70% ethanol, 3 mL of distilled water, and then 3 mL of I-solution at a flow rate of 300 µL/s.
3. Warm the C- and I-solutions to 37 °C in the water bath.
   NOTE: As 100 µL of each solution is needed for each well of the 96-well plate, about 10 mL of each solution is needed for each assay. An additional 10 mL of the I-solution is needed for priming each plate (total of 20 mL) and an additional 25 mL of the C-solution is needed for washing each plate (total of 35 mL).
4. Aspirate the growth medium or invert the plate to empty it; tap out residual medium.
   NOTE: Residual fetal bovine serum in the growth media causes background fluorescence and a decline in assay quality.
5. Add 200 µL of C-solution to each well from a reservoir using a multichannel pipette. Aspirate the C-solution or invert the plate to empty and tap out residual solution.
6. Add 50 µL µL C-solution, 1 µL of 2.5 mM chemical stock (see Table 1) or dimethylsulfoxide (DMSO) as a vehicle, and then 50 µL µL C-solution to each well with a multichannel pipette.
   NOTE: Most reagents in a chemical library are dissolved in DMSO and up to 1% (v/v) is allowed in most cell-based assays²⁴. As DMSO has a higher density than water, reagents dissolved in DMSO tend to go down to the bottom when added to the culture plate wells, which disturbs the concentrations of the assay solutions. This can be circumvented by adding 50 µL of C-solution, reagents in DMSO, and 50 µL C of -solution in order. The last 50 µL of C-solution is for mixing.
7. Incubate the cells at 37 °C in air, not in 5% CO₂. The incubation time can be modified, but 10 min is usually sufficient for ion-channel modulators to act.
8. During incubation, set the microplate reader program to inject 100 µL of I-solution to each well at 1 s and to measure the fluorescence for 10 s at 0.4 s intervals. Set the reader to read fluorescence from the bottom. The recommended injection speed is 135 µL/s. Set the excitation wavelength to 485 nm and read the emission at 520 nm.
   NOTE: Detailed settings for the microplate reader programs are as follows.
   1. Click the Manage protocols button.
   2. Click the New Button. Select the Fluorescence Intensity in the measurement method section, and Well Mode in the reading mode section. Next, click the OK button. New tab will appear.
   3. In the Basic Parameters menu, set the excitation wavelength to 485 nm and the emission at 520 nm. Select the Bottom Optic to read fluorescence from the bottom. Set measurement start time to be "0 s", number of intervals to be "25", number of flashes per well, and interval to be "20", and interval time to be "0.4 s".
   4. In the Layout menu, draw region of the plate to be read.
   5. In the Concentrations/Volumes/Shacking menu, set the microplate reader to inject 100 µL of I-solution to each well with 135 µL/s injection speed.
      NOTE: Avoid faster injection speeds because they can result in detachment of cells.
   6. In the Injection time menu, set the injection start time to be 1 s. Then, click the Start Measurement button.
9. After incubation, place the 96-well plates in the microplate reader and start the measurement by clicking Start Measurement button again.

6. Calculation of GJIC activity

1. Calculate the percentages of YFP^QL quenching and GJIC activity as²³
   YFP^QL quenching (%) =
   GJIC activity (%) =
   NOTE: In principle, GJIC activity should be calculated from the difference of the percentages of YFP fluorescence in wells with acceptor cells and donor cells and the corresponding acceptor-cell only wells. However, as LN215-YFP^QL cells show negligible YFP quenching by iodide after 10 s, we do not take the background YFP quenching into account when conducting HTS using LN215-YFP^QL and LN215-I^- cells.

Results

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-YFP^QL 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...

Discussion

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...

Materials

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.G	Addgene	#12259
Polybrene	sigma	H9268
Poly-L-lysine solution	sigma	P4707
Potassium chloride (KCl)	Sigma	P5405	C-solution, I-solution
psPAX2	Addgene	#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