In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

1. Construction of PER2 Promoter Driven Destabilized Luciferase Reporter Vector

1. Purchase a customized pLS[hPER2P/rLuc/Puro] vector that contains cDNA encoding rLuc and human PER2 promoter fragment (hPER2P, 941 bp) at the site between Sac I and Hind III in the multiple cloning region¹⁷.
2. Cut the human PER2 promoter fragment (hPER2P, 941 bp) out from the vector.
   1. Add 2.5 µL restriction enzyme buffer, 10 µL (180 ng) pLS[hPER2P/rLuc/Puro] vector, and 1 µL each of the restriction enzymes, Sac I (10 unit/µL) and Hind III (10 unit/µL), into a DNA/RNA/nucleotide-free microcentrifuge tube. Add ultrapure water (10.5 µL) up to 25 µL and mix gently by pipetting.
   2. Incubate at 37 °C for 90 min in a heating block.
   3. Run the whole volume (25 µL) of the restriction enzyme reaction on 0.7% agarose gel containing 0.0001% ethidium bromide (EtBr) to separate the hPER2P from the vector.
      NOTE: EtBr, 3,8-diamino-1-ethyl-6-phenylphenantridinium bromide (CAS registration number: 1239-45-8), is an odorless red liquid. It is an intercalating agent that is commonly used as a fluorescent tag (nucleic acid stain) in agarose gel electrophoresis and visible as an orange color under UV light. Possible risks of irreversible mutagenic effects have occurred in experimental animals, although none of the regulatory agencies categorized it as a carcinogen¹⁸. Therefore, we collected used agarose gel and electrophoresis buffer containing EtBr as chemical hazard waste, and requested Rutgers Environmental Health & Safety (REHS) to pick up and dispose safely.
   4. Cut a piece of agarose gel containing a EtBr fluorescence band at 941 bp, and purify the hPER2P fragments from the gel with a DNA gel extraction kit, followed by quantification on a spectrophotometer by measuring absorption density (OD) at 260 nm wavelength.
3. Linearize 1.0 µL (1 µg) of the destabilized firefly luciferase expression vector, pGL[Luc2P/Neo] with the same method as described in steps 1.2.1-1.2.2. Extract the vector with a DNA clean-up kit followed by quantification as described above using a spectrophotometer.
4. Ligate the hPER2P into the linearized vector pGL[Luc2P/Neo].
   NOTE: Per the manufacturer's instruction, the ideal molar ratio of insert and vector is 2:1. For 50 ng vector, the ideal amount of insert is calculated as 23.6 ng with a formula, [(50 ng vector x 1.0 kb insert)/4.242 kb vector] x (2/1) = 23.6 ng insert]. Based on the concentrations of hPER2P (7.26 ng/µL) and pGL[Luc2P/Neo] (50 ng/µL), the volumes of 50 ng pGL[Luc2P/Neo] and 23.6 ng hPER2P are calculated as 1 µL and 3.26 µL, respectively.
   1. Add 2 µL T4 DNA ligase reaction buffer (10X) (50 mM Tris-HCl, 10 mM MgCl₂, 1 mM ATP, and 10 mM DTT), 3.26 µL (23.6 ng) hPER2P, 1 µL (50 ng) pGL[Luc2P/Neo], and 13.74 µL water up to 20 µL, mix gently.
   2. Add 1 µL T4 DNA ligase (400 unit/µL), mix gently and incubate at 16 °C overnight in a thermocycler, and then chill the ligated vector on ice per the manufacturer's instruction.
5. Transform the ligated vector pGL[hPer2P/Luc2P/Neo] to chemically competent E. coli¹⁹.
   1. Set the water bath at 42 °C, warm up Super Optimal Broth with catabolite repression (S.O.C.) medium (2% Tryptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, and 20 mM glucose) at room temperature, warm up Luria-Bertani (LB) agar plate (containing 100 µg/mL ampicillin, 80 µg/mL X-gal, and 0.5 µM IPTG) in 37 °C incubator, and thaw on ice one vial of competent E. coli cells for each transformation.
   2. Add 6 µL (21 ng) ligated vector or 1 µL (30 ng) empty vector (negative control) to one tube of chemically competent E. coli (50 µL) and then gently flip the tube to mix. Incubate on ice for 30 min.
   3. Heat shock in a 42 °C water bath for 30 s without shaking and then return to ice.
6. Select an E. coli colony transformed with a ligated vector using blue/white screening.
   1. Add 250 µL S.O.C. medium in the transformed E. coli tube and incubate it for 1 h at 37 °C with shaking at 200 rpm.
   2. Spread 10-50 µL of transformed E. coli on the surface of the LB agar plate evenly. Put the plate upside down in 37 °C incubator overnight.
      NOTE: An efficient cloning reaction should produce several hundred colonies. Plating two different volumes is recommended to ensure that at least one plate will have big, clear white or blue color, and well-spaced colonies. When the colonies are too small and the color is not distinguishable, using a microscope (10X or 50X) is helpful.
   3. Pick 3-6 white colonies from the plates with sterilized tooth sticks, and release each colony into one culture tube containing 4 mL of LB culture medium with 50 µg/mL ampicillin.
   4. Incubate the tubes at 37 °C with shaking at 200 rpm overnight. Extract the DNA using 2 mL of the 4 mL cultured E. coli with a plasmid DNA extraction mini prep kit per the manufacturer's instruction.
7. Verify and analyze the insert (hPER2P, 941 bp)
   1. Digest the plasmid DNA with restriction enzymes and run electrophoresis as described in steps 1.2.1-1.2.3 to confirm if there is an insert.
      NOTE: Positive control (hPER2P purified at step 1.2.4) and negative control (E. coli transformed with an empty vector) were included.
   2. Sequence the selected plasmid DNA using M13 forward and M13 reverse primers to confirm the orientation and sequence of insert in the plasmid¹⁹.
      NOTE: Only one colony that had an insert with the correct size of hPER2P in electrophoresis and correct orientation and sequence in the sequencing result was selected.
   3. Analyze the human PER2 promoter fragment (hPER2P, 941 bp) sequence for the circadian regulatory elements, including E-box motif (Bmal1 binding site, CAT/CGTG), CCAATC, GC box, and transcription start site (CAGCGG)²⁰.
8. Amplify the selected E. coli by adding the leftover 2 mL cultured E. coli into 200 mL LB culture medium containing 50 µg/mL ampicillin and then incubating it overnight at 37 °C with shaking at 200 rpm.
9. Extract DNA with an endotoxin-free plasmid maxi prep kit per the manufacturer's instruction, and then quantify it by measuring OD at 260 nm wavelength with a spectrophotometer. Aliquot and store the plasmid DNA, pGL[hPer2P/Luc2P/Neo], at -80 °C freezer for future use.

2. Transient Transfection

1. Purchase and culture MCF10A cells
   1. Purchase an immortalized, non-transformed normal human mammary epithelial cell line (MCF10A) from a commercial cell bank, where cells are cytogenetically tested and authenticated with short tandem repeat analysis before freezing. Thaw and maintain each vial of frozen cells in culture for a maximum of 8 weeks.
      NOTE: Unlike other cells, these cells have contact inhibition once they get to ~70% confluence. It requires that subculture is conducted at ~70%.
   2. Culture the cells with mammary epithelial cell growth medium (MEGM), containing mammary epithelial basal medium (MEBM), growth supplements, and cholera toxin in a cell culture incubator at 37 °C, 95% humidity, and 5% CO₂.
      NOTE: Purchase ready-to-use growth factors provided in SingleQuot (SQ) and obtain cholera toxin separately. Final concentrations of growth supplements are 0.4% bovine pituitary extract, 0.1% insulin, 0.1% hydrocortisone, 0.1% human epidermal growth factors, 100 ng/mL cholera toxin, and 0.05% gentamycin sulfate and 0.05% amphotericin B.
   3. Dissociate the cells in 10-cm culture dish by incubation with 10 mL trypsin/EDTA for ~ 20 min and then neutralize the trypsin by adding 10 mL trypsin neutralizing solution. Collect all cells with HEPES buffered saline solution (HBSS).
   4. Count the cells with a hemocytometer under an inverted microscope and calculate the concentration of the single cell suspension, and then seed a certain number of cells as needed. Swirl the plates thoroughly to obtain an even distribution of cells in each plate. Incubate cells in the cell culture incubator at 37 °C, 95% humidity, and 5% CO₂.
      NOTE: Purchase a subculture reagent pack containing trypsin/EDTA, trypsin neutralizing solution, and HBSS to use.
2. Transient transfection of cells with the circadian vector constructed.
   1. Seed 2 x 10⁵ MCF10A cells evenly in 35 mm culture dish with MEGM and grow to 20-30% confluence (next day).
   2. Warm up the reduced serum media (e.g., Opti-MEM) in 37 °C water bath and transfection reagent at room temperature for 30 min. Dilute the plasmid DNA constructed (pGL[hPer2P/Luc2P/Neo]) to 30 ng/µL with endo-toxin free Tris-EDTA (TE) buffer and keep at room temperature.
   3. Prepare the plasmid transfection mixture in a 1.5 mL microcentrifuge tube by adding 63.6 µL warm reduced serum media first, then adding 33.4 µL (0.1 µg) plasmid DNA, and mix gently by pipetting, and then lastly adding 3 µL of transfection reagent directly into the center of the tube without touching the wall. After mixing gently by pipetting, keep at room temperature for 30 min.
   4. Drop the entire plasmid mixture (100 µL) directly onto the center surface of the medium in the plate and then mix gently by shaking the dish. Culture the cells in a CO₂ incubator for additional 48-72 h.
      NOTE: It is predicted that the highest transfection efficiency would be at 40-70% confluence of these cells due to their contact inhibition at ~ 70%. Therefore, transfection works best starting at ~ 20% and stop at ~ 70% confluence.

3. Establishment of the In Vitro Bioluminescence Assay in Transiently Transfected MCF10A Cells

1. Starve the cultured cells at ~70% confluence (after transfection for 48-72 h) in MEBM without growth factors for 24 h.
2. Treat the cells with synchronization agent (e.g., 50% horse serum (HS)) in MEBM for 1.5 h in a CO₂ incubator.
   NOTE: For selection of an ideal synchronization agent, 10 µM forskolin, 1 nM melatonin, 0.1 µM dexamethasone, 50% HS, and 100% SQ were compared in terms of circadian amplitude and period in the circadian vector transfected cells. Empty vector transfected cells are treated with 100% SQ as a negative control.
3. Pre-prepare 20 mL recording medium (for 10 of the 30-mm culture dishes) by adding 5 mL of MEGM, 15 mL of MEBM, 150 µL sodium bicarbonate, 200 µL HEPES, and 40 µL luciferin stock solution (50 mM) in a 50 mL sterilized centrifuge tube.
   NOTE: The final concentrations of each components: 20% SQ and 20 ng/mL cholera toxin, 0.06% sodium bicarbonate, 1% penicillin/streptomycin, and 10 mM HEPES. Warm up pre-prepared recording medium and sterilized 1x PBS for 30 min in 37 °C water bath. Add 100 µM luciferin immediately before use.
4. Wash the cells with warm 1x Dulbecco's Phosphate-Buffered Saline (D-PBS) for 3 times after the incubation with the synchronization agent, and then add 2 mL recording medium.
5. Seal the dish with a sterilized cover class using silicon grease to prevent evaporation and then label it on the side.
6. Place the sealed dish in the seat inside the luminometer, and turn on the option for saving the file location in the folder (for the detail, see section 6).

4. Selection of Stably Transfected Cells

1. Optimize an ideal G418 concentration (800 µg/mL) with a kill curve assay according to the manufacturer's instruction for selection of the stably transfected cells.
2. After transient transfection for 48 h or 72 h, subculture and seed the cells with MEGM in larger dishes at 5,000 cells/dish (100-mm). After 24 h culture in the cell culture incubator (37 °C, 95% humidity, and 5% CO₂), treat the cells with 800 µg/mL of G418 by replacing the old medium with new medium containing 800 µg/mL G418 every 3-4 days for 2 weeks.
3. Subculture the surviving stably transfected colonies for 1-3 passages with MEGM containing 500µg/mL G418 to maintain the stable expression of hPer2P/Luc2P, and freeze in liquid nitrogen for future use.
4. Treat 500µg/mL G418 after general subculture to re-select the stably transfected population of cells, if the bioluminescence intensity in the result of in vitro bioluminescence assay becomes progressively lower after use in many passages.

5. Treatment with Chemicals

1. At 48 h or 72 h post-transfection, starve the cells from growth factors in MEBM for 24 h.
2. After synchronization with 50% HS for 2 h, treat cells with 0.25 mM or 0.5 mM nitrosomethylurea (NMU), 20 nM EX527, or 1 µM cambinol for 1 h in MEBM containing 20% SQ.
3. After washing with 1x D-PBS, add recording medium containing 12.5 µM MSC alone, or in combination with 20 nM EX527 or 1 µM cambinol, to the cells. Monitoring the bioluminescence with the luminometer.
4. Treat the stably transfected MCF10A/PER2–dLuc cells with NMU at 0.5, 1, or 2 mM, EX527 at 40 nM, or Cambinol at 2.0 µM, for 1 h after synchronization with 50% HS, and then incubate the cells in recording medium containing 12.5 µM of MSC or different doses (5, 10, or 20 µM) of n-acetylcysteine (NAC). Place the plates in the luminometer, record and save the bioluminescence by luminometer for 4-8 days as described in section 3.6.
   NOTE: NMU is a methylated nitrosourea compound with mutagenic, carcinogenic, and teratogenic properties. NMU is a direct-acting alkylating agent and has a short half-life (T_1/2 = ~ 30 min). It is stable at acidic condition (pH 4-5), but unstable at alkaline condition (pH 9-10) and at temperatures beyond 20 °C. Therefore, bleach at 10% can be used as an efficient deactivating agent for cleaning bench surfaces and lab wares potentially contaminated by NMU solution. Leftover stock solution is picked-up and safely disposed of by REHS. Based on the mode of action of chemicals, synchronized cells can be treated for shorter times before culturing in recording medium. Cells can also be treated in the recording medium for a longer time (several days).

6. Data Collection, Analysis, and Presentation

NOTE: Cell viability needs to be determined using standard methods (e.g., MTT assay) after treatment of cells at the same concentrations for the same times indicated. All treatment concentrations used in the in vitro bioluminescence assay were lower than the 30% lethal concentration (LC30). All experiments were conducted in triplicate and representative results were presented.

1. After sealing the dish (step 3.5), load the dishes onto the seats in the luminometer, which is kept inside an incubator set at 37 °C without H₂O and CO₂, and connected to a computer.
2. Click "save" and enter the names for the folder and file where the recorded luminescence signal from the corresponding dish will be saved.
   NOTE: The system detects the luminescence signal in real-time from the cells in the dish at individual positions. The signals are transferred to the computer through 4-photon-counting photomultiplier tubes, and saved and displayed in the computer by the data collection software.
3. Analyze the signals after recording for 4-7 days, which could be followed by medium change and continuous recording for a second week if necessary.
   NOTE: To obtain circadian parameters, including phase, period length, rhythm amplitude, and damping rate, we used the luminometer analysis software to analyze the bioluminescence data.
4. Detrend raw data with a running average, and analyze the best-fits to a sine wave to get period, phase, amplitude, and damping rate^21,22.
5. Due to the high transient bioluminescence upon treatment and medium change, exclude the first cycle of data from analysis.
6. For data presentation, plot raw data (bioluminescence, count/s) against time (h or day). When necessary, baseline-subtracted data can be plotted to compare amplitude and phase.