Materials and Reagents

Pipette tips (USA Scientific, catalog number: 1111-1700 )
100-mm tissue culture grade plastic plates (SARSTEDT, catalog number: 83.3902 )
5 ml tissue culture tubes (Corning, Falcon^®, catalog number: 352052 )
Cell culture flasks (Corning, catalog number: 3056 )
96-well OptiPlates (PerkinElmer, catalog number: 6005290 )
15 ml conical tube (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 339650 )
Pasteur pipette (Fisher Scientific, catalog number: 13-678-20B )
Rapid flow bottle-top filter unit with polyethersulfone (PES) membrane (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 595-3320 )
PS1/PS2-deleted HTL cells
Note: This cell line was derived by Xu et al., 2016, utilizing CRISPR/Cas9 to delete PS1/PS2 from HTL cells, representing a cell line derived from HEK293 cells by Barnea and Axel who stably integrated a luciferase reporter under the control of the bacterial operator element tetO. These cells were used in this case to correlate with specific functional assays requiring a specialized cell type. In a general protocol, any transfectable cell type is fine.
Dulbecco’s modified Eagle’s medium powder (DMEM) (Thermo Fisher Scientific, Gibco^TM, catalog number: 12100-038 ) (see Recipe 6 for media preparation)
Fetal Clone II supplement, a bovine serum product (GE Healthcare, Hyclone^TM, catalog number: SH30066.03 )
Trypsin 0.25%-EDTA (Thermo Fisher Scientific, Gibco^TM, catalog number: 25200056 ) diluted to 0.05% with 1x Dulbecco’s phosphate buffered saline, pH 6.80
DEAE-dextran hydrochloride (Sigma-Aldrich, catalog number: D9885 )
Dimethyl sulfoxide (DMSO) (Fisher Biotech, catalog number: BP231-1 )
Chloroquine diphosphate salt (Sigma-Aldrich, catalog number: C6628 )
Enzyme-free cell dissociation solution (EMD Millipore, catalog number: S-014-C )
Sodium phosphate dibasic (Na₂HPO₄) (Sigma-Aldrich, catalog number: S0876 )
Potassium phosphate monobasic (KH₂PO₄) (Fisher Scientific, catalog number: P285 )
Sodium chloride (NaCl) (Fisher Scientific, catalog number: S671-500 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
HEPES (Research Products International, catalog number: H75030-1000 )
Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (Sigma-Aldrich, catalog number: M1880 )
Calcium chloride dihydrate (CaCl₂·2H₂O) (Fisher Scientific, catalog number: C79-500 )
Sodium hydroxide (NaOH) (VWR, catalog number: BDH7247-1 )
Coelenterazine-h (AAT Bioquest, catalog number: 21165 ) (see Recipe 5 for preparation of stock solution)
Methanol (Honeywell International, catalog number: 24229 )
Penicillin-streptomycin (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
Sodium bicarbonate (Fisher Scientific, catalog number: BP328-500 )
Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A144-212 )
10x phosphate-buffered saline (PBS buffer) (see Recipes)
10x Kreb’s-Ringers-HEPES stock solution (10x KRH) (see Recipes)
50x CaCl₂ stock solution (see Recipes)
1x KRH working solution (see Recipes)
Coelenterazine-h solution (see Recipes)
1x DMEM tissue culture medium (see Recipes)

Equipment

37 °C, 5% CO₂ incubator (Thermo Fisher Scientific, Thermo Scientific^TM, model: Forma^TM Series II 3110 Water-Jacketed)
Cell culture microscope (Fisher Scientific)
Micropipettes (Gilson)
2103 EnVision Plate Reader (PerkinElmer, model: 2103 EnVision^TM )
Centrifuge (Thermo Fisher Scientific, Thermo Scientific^TM, model: Sorvall^TM Legend^TM XT/XF centrifuge , catalog number : 75216362)
500 ml screw-cap autoclaved glass bottles (WHEATON, catalog number: 219759 )

Software

GraphPad Prism 6 (GraphPad Software, Inc. USA)

Procedure

Cell culture
1. The PS1/PS2-deleted HTL cells are routinely grown in DMEM medium supplemented with 10% (v/v) Fetal Clone II and including 1x penicillin-streptomycin (see Recipes) at 37 °C under a humidified 5% CO₂ atmosphere.
2. Treat the cells with 0.05% trypsin-EDTA for 5 min at 37 °C in an incubator to lift the cells. Wash the cells with medium, and count them to allow a plating density of 1.5 x 106 cells per 100 mm dish one day prior to transfection.
3. In preparation for transfection, visualize the confluence of the cells and their health using a cell culture microscope with phase optics.
Transfection
1. To a sterile 5 ml tissue culture tube, add 1.5 ml DMEM medium without serum.
2. Add 75 µl DEAE-dextran solution and mix well.
3. Add 1.0 µg of total DNA (0.5 µg of donor + 0.5 µg of acceptor prepared using standard DNA isolation techniques) and mix well.
4. Use a micropipette to transfer 1.575 µl of DNA-DEAE-dextran complex to each plate.
5. Incubate at 37 °C in a tissue culture incubator for 2 h without shaking.
6. Aspirate the medium from each plate, add 4 ml of DMEM containing 10% DMSO and incubate at room temperature for 2 min without shaking.
7. Aspirate the medium from each plate, add 4 ml of DMEM containing 0.1 µM chloroquine and incubate for 2 h at 37 °C without shaking.
8. Aspirate the medium from each plate, and add 8 ml of serum-containing complete DMEM medium (see Recipes).
9. Incubate the cells in the incubator at 37 °C in a humidified atmosphere containing 5% CO₂ for 48 h.
Luminescence and fluorescence measurement
1. 48 h post-transfection, remove the medium from the cultured cells using a Pasteur pipette attached to a vacuum source and gently add 5 ml of PBS (see Recipes) to each plate to rinse the cells.
2. Dispense 1 ml of enzyme-free cell dissociation solution into each plate and incubate without shaking for 5 min at 37 °C in tissue culture incubator to release the cells.
3. Transfer the cells into a 15 ml conical tube and centrifuge the cells at 175 x g for 5 min at room temperature.
4. Resuspend the cell pellet in approximately 1 ml of Kreb’s-Ringers-HEPES medium, pH 7.4 (see Recipes) to achieve a density of 0.20 x 10⁶ cells per ml. Keep this tube at room temperature in preparation for the assay.
5. Dispense 100 µl of cell suspension (approximately 20,000 cells) into a white 96-well OptiPlate, add 5 µl coelenterazine h (see Recipes), mix the cells by swirling the plate, and measure both the luminescence signal and the YFP fluorescence signal using an Envision Plate Reader with a BRET module.
6. Luminescence and fluorescence signals should be collected immediately after the addition of coelenterazine h solution. The spectrofluorometer instrument should be set up for the specific pair of donor and acceptor chosen. Here, for the proposed BRET studies, luminescence emission should be collected using a dedicated filter set (460 nm, with bandwidth 25 nm) and the fluorescence emission should be collected simultaneously using a dedicated filter set (535 nm, bandwidth 25 nm). The measurement time is 0.2 sec and measurement height is 6.5 mm. (Harikumar et al., 2007). In the example, YFP fluorescence was measured by exciting the sample at 475 nm (bandwidth 25 nm) and the emission was collected at 535 nm (bandwidth 25 nm), with detector gain 150 and number of flashes 20.
7. A representative example of data collection and analysis follows:
  
  Calculated BRET ratio: 1.23 - 0.70 = 0.53

Data analysis

Data should be collected in at least duplicate conditions in a minimum of three independent experiments. The statistical analysis of data can be accomplished with GraphPad Prism 6, using a t-test with the Mann-Whitney post-test.

Notes

Controls for levels of expression of donor and acceptor are important to eliminate bystander energy transfer. A significant BRET signal should be observed at levels of expression that exist physiologically. Additionally, saturability and specificity of the BRET signal are important, and can be established using competing non-labeled protein of interest. For positive control, a construct encoding a fusion protein containing both luciferase and yellow fluorescent protein (Rlu-YFP) was used to transfect the cells, and the BRET signal was measured.

Recipes

10x phosphate-buffered saline (10x PBS buffer) (1 L)
11.5 g Na₂HPO₄
2 g KH₂PO₄
80 g NaCl
2 g KCl
Dissolve in 1 L of sterile, deionized water
Adjust the pH of 1x PBS to 7.4
Store at 4 °C
10x Kreb’s-Ringers-HEPES stock solution (10x KRH) (1 L)
59.58 g HEPES
60.74 g NaCl
3.72 g KCl
1.36 g KH₂PO₄
2.96 g MgSO₄
Dissolve the components in deionized water and bring the volume to 1 L, do not adjust the pH
Store at 4 °C
50x CaCl₂ stock solution (100 mM) (100 ml)
1.47 g CaCl₂
Bring the volume to 100 ml with deionized water; do not adjust pH
Store at 4 °C
1x KRH working solution (100 ml)
10 ml 10x KRH stock solution
2 ml 50x CaCl₂ stock solution
Bring the volume to 100 ml with deionized water; adjust the pH to 7.4 with NaOH
Coelenterazine-h solution
0.5 mg coelenterazine-h powder
Dissolve in 12.5 ml of 5% methanol/95% water
Store at -20 °C
1x DMEM tissue culture medium (1 L)
1. One 1-L packet of Dulbecco’s modified Eagle medium (13.4 g) and 3.7 g of sodium bicarbonate are added to 1 L of deionized water
2. Adjust the pH to 6.90 with HCl, filter into 500 ml screw-cap autoclaved glass bottles using a 0.2 µm rapid flow bottle-top filter unit containing a polyethersulfone (PES) membrane
3. Store at 4 °C

Acknowledgments

This work was supported by Mayo Clinic (L.J.M.), the Van Andel Research Institute, the National Natural Science Foundation of China (31300607, 31300245 and 91217311), Ministry of Science and Technology grants 2012ZX09301001, 2012CB910403, and 2013CB910600, XDB08020303, 2013ZX09507001, Shanghai Science and Technology Committee (13ZR1447600), Shanghai Rising-Star Program (14QA1404300), and the National Institute of Health grants DK071662 (H.E.X.), GM102545 and GM104212 (K. M.). Authors have no conflicts of interest or competing interests.

References

Dacres, H., Wang, J., Dumancic, M. M. and Trowell (2010). Experimental determination of the Förster distance for two commonly used Bioluminescence resonance energy transfer pairs. Anal Chem 82: 432-435.
Harikumar, K. G., Pinon, D. I. and Miller, L. J. (2007). Transmembrane segment IV contributes a functionally important interface for oligomerization of the Class II G protein-coupled secretin receptor. J Biol Chem 282: 30363-30372.
Xu, T. H., Yan, Y., Kang, Y., Jiang, Y., Melcher, K. and Xu, H. E. (2016). Alzheimer's disease-associated mutations increase amyloid precursor protein resistance to γ-secretase cleavage and the Aβ42/Aβ40 ratio. Cell Discov 2: 16026.