Template:UniGE-Geneva/Protocols

Unige iGEM 2019

Protocols

Monolayer cell co-culture

Estrogen deprivation

Aims:

Preparation of cells to remove all trace of estrogens in the culture medium before the induction of cells by estrogen treatment.

Materials:

HepG2 cells (Hepatic carcinoma cell line) and MELN cells (MCF-7 cell line stably expressing luciferase under the control of an Estrogen Response Element), 10 cm-dishes, White DMEM medium: DMEM without phenol red, complemented with 5% charcoal-stripped serum, 2mM L-Glutamin and Penicillin/Streptomycin, Coverslips 12 mm of diameter, CO2 Incubator

Procedure:

Charcoal-stripped serum is prepared by incubating Fetal Bovine Serum (FBS) with 2% activated charcoal for 30 minutes. FBS is then centrifugated at 8,000 rpm for 10 minutes to remove activated charcoal in the pellet. This step is repeated two more times. After the final centrifugation, FBS is collected and filtrated through a 0.2µm filter. Cells were seeded in separated dishes, with 3 million cells/dish in white DMEM medium. Coverslips were put at the bottom of the dishes to allow cells to grow on them. Cells were grown at 37°C for 4 days.

Induction of Estrogen Receptor

Aim:

To induce estrogen receptor in the presence or not of increasing concentrations of pharmacological inhibitors to make a dose-response curve.

Materials:

6 well-plates, White DMEM medium (for composition, see “Estrogen deprivation” section), Ethanol 100%, 17 beta-estradiol 10nM, 4-hydroxytamoxifen (concentration range between 100nM and 1mM), Tamoxifen (concentration range between 30nM and 1mM), CO2 Incubator

Procedure:

Coverslips seeded 4 days before were transferred into 6 well-plates in 2 ml of DMEM medium per well. We did two different combinations of coverslips per well: two coverslips with MELN cells (1), or one coverslip with MELN cells and one coverslip with HepG2 (2). Treatments were performed in triplicate for each condition. All molecules are diluted in 100% Ethanol at a concentration 1000-fold higher than their final concentration in the cell culture medium: cells were treated with a total volume of 4 µl of molecule per well (2 µl of estrogen (E2) + 2 µl of inhibitor, or 2 µl of estrogen (E2) + 2 µl of Ethanol for the control). Treatments are homogenized by rotating the plate right after putting the molecule.

The different treatment were as followed, with the final concentrations:

- Ethanol, 17 beta-estradiol (E2) 10 pM

- E2 10 pM + 4-hydroxytamoxifen (Dose-response: 1µM, 300 nM, 100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 300 pM)

- E2 10 pM + tamoxifen (Dose-response: 1µM, 300 nM, 100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 300 pM).

After treatments, cells were incubated at 37°C for 2 days.

Measurement of Luciferase activity

Aim:

Luciferase activity measurement reports the activity of Estrogen Receptor in MELN cells depending on the different treatments and combinations of co-culture.

Materials:

24 well-plates, White DMEM medium, Passive lysis buffer (promega), Phosphate buffer saline (PBS), Rotating shaker, White 96 well-plates, Luciferase substrate (Promega dual-luciferase assay kit)

Procedure:

Coverslips of MELN cells were transferred into a 24 well-plate filled with 1 ml of DMEM medium in each well to avoid cells drying during the transfer process. After the transfer of coverslips, medium was aspirated and cells were lysed with 50 µl of Passive Lysis Buffer 5X diluted 20 times in PBS. Plates were incubated at room temperature for 10 minutes on a rotating shaker at 100 rpm. White 96well-plates were loaded with 10 µl of Luciferase substrate and then 10 µl of lysates were added to each well. Luciferase activities were measured for each well with a luminometer to quantify the bioluminescence.

Encapsulation protocol

Preparation of alginate

Aim:

Preparation of alginate and fluorescentl-labeled alginate to make different types of capsule.

Materials:

Alginate, Fluorescently-labeled alginate stock solution (see “fluorescent labeling of alginate” section), ddH20, rotator

Procedure:

Unlabeled alginate: Prepare a solution of alginate 2% in a falcon of 10ml or 50ml (depending of the amount needed). 0.2g of alginate for 10ml of ddH20. Mix the solution overnight on a rotator at room temperature.

Fluorescently-labeled alginate: Prepare a solution of alginate 2% in a falcon of 10ml or 50ml (depending of the amount needed). Add a volume of stock solution of fluorescently-labeled alginate (see “Fluorescent labeling of alginate” section) in the solution of alginate with a ratio labeled:unlabeled alginate of 1:10. Mix the solution overnight on a rotator at room temperature.

Preparation of cells for encapsulation

Aim:

Preparation of cells that will be encapsulated into an alginate capsule.

Materials:

Red DMEM medium: DMEM supplemented with 10% Fetal Bovine Serum and Penicillin/Streptomycin, Trypsine, Tris buffer saline (TBS), 40µm filters, Sorbitol

Procedure:

Cells were washed with TBS, trypsinized, and collected in red DMEM medium. Cell suspension was passed through a 40µm filter in a 50ml Falcon tube to discard cell aggregates that could plug the microfluidic chip. Cells were counted to prepare Eppendorf tubes with 4 million of cells. Cells were centrifuged at 1000 rpm for 5 minutes to pellet them and they were resuspended in Sorbitol (90µl). Cells were kept on ice until encapsulation.

Encapsulation

Aim:

To encapsulate cells into an alginate capsule.

Materials:

Microfluidic device, Sorbitol, Alginate, Cell suspension, CaCl2 buffer, Tween20, 18-gauge needles, Red DMEM medium, Red DMEM medium complemented, CO2 Incubator

Encapsulation procedure:

All the syringes of the microfluidic device were prepared with specific solutions (two syringes filled with sorbitol and one with alginate). Cell suspension is placed in a pipe connected to one of the syringes of sorbitol. The three syringes are then connected to the chip. The pump of the device is turned on to inject the different solution in the different pipes lastly connected to the microfluidic chip. When the cell suspension reaches the chip, a ring of electric field is turned on to avoid the formation of alginate clusters. Droplets falling down from the chip were collected directly into a plate filled with a solution of CaCl2 0.1 M + Tween20 (to avoid viscosity) and starts to harden to form the capsule. Alginate capsule were incubated 30 min at room temperature in the CaCl2 solution to finish to harden.

Procedure after the encapsulation:

Calcium chloride solution is aspirated incompletely (by letting about 10ml of liquid) with 18-gauge needles by staying as close as possible to the surface to avoid to aspirate the capsules. The dish is refilled with 60ml of red DMEM medium not complemented. After waiting that the capsules are readily at the bottom of the dish, the medium is again aspirated incompletely, and the step of refilling and aspirating is repeated two more times. Fill the dish with 20 ml of complete red medium that contains usual concentration of serum, aspirate incompletely, refill again with 20ml of complete red DMEM medium and put the dish with the capsules at 37°C.

Test of reporter protocol

Cell transfection

Aim:

To introduce transiently into human cells DNA reporter constructs (standard protocol for all the reporter constructs tested during the project).

Materials:

CO2 Incubator, White DMEM medium, 24-well plates, Plasmids, Tris buffer saline (TBS), Polyethylenimine (PEI), HEK293T cells, Trypsin

Procedure:

HEK293T cells already deprived in white DMEM medium (see protocol “Monolayer cell co-culture”, section “Estrogen deprivation”) during 4 days are collected by trypsinization and seeded in 24-well plates at 80,000 cells/well in 0.5 ml of DMEM medium. Incubate the cells at 37°C for 24 hours.

Master mix (transfection solution) - calculation for one well:

- 1µg of DNA was added to 12.5µl of TBS and vortexed. Many different combinations of plasmids were used depending on the reporter construct that was tested.

- 3µl of PEI (PolyEthylenImine) Max are added to the tube and immediately mixed by pipetting up and down several times, followed by vortexing.

Incubation of 15 minutes at room temperature. Then the well is transfected by putting directly 12.5µl of the transfection solution (master mix) directly on the cells and immediately mixed by rotating the plate. Incubation at 37°C overnight.

Induction of reporter constructs

Aim:

Induction of the reporter constructs with their specific inducer.

Materials:

White DMEM medium, Specific inducer, CO2 incubator

Procedure:

After overnight incubation with the transfection mixture, the medium is replaced with 1ml of fresh white DMEM medium per well. In the afternoon, cells are treated with the appropriate inducer: FBS 15% for the FUCCI reporter, Staurosporine 1µM for the zip-GFP reporter, and E2 100nM for the ERE-SYFP2 reporter that we designed for the iGEM competition. Incubate with the treatments overnight at 37°C.

Measurement of the fluorescence of reporters

Aim:

Quantification of the fluorescence intensities of the reporters to determine their levels of induction.

Materials:

Phosphate buffered saline (PBS), White 96 well-plates, Microplate multi-reader (Cytation)

Procedure:

Aspirate the medium of the HEK293T cells and fastly put 200 µl of PBS. Pipetting up and down to detach the HEK293T cells (these cells can detach without trypsinization). Collect 150 µl of the cell suspension and transfer it in a white 96 well-plate. Read the fluorescence intensities with the microplate multi-reader with the proper excitation and emission wavelengths for each fluorescent reporter that was tested.

Cloning protocol

Digestion of destination vector

Aim:

To cut the destination vector (pHAGE-fEF1a-zsGreen) for the cloning of inserts.

Materials:

pHAGE vector, Restriction enzyme buffer, Restriction enzymes SpeI and XhoI (NEB), Heat block

Procedure:

Mix together in a tube: 5µg of pHAGE vector, 5µl of restriction enzyme buffer 10X, 10U of SpeI, 10U of ClaI, Up to 50µl nuclease-free water. Incubation 3 hours at 37°C followed by heat inactivation at 80°C for 20 minutes.

Amplification and modification of inserts

Aim:

Amplification of insert with specific primers and insertion of modifications at the 5’- and 3’-ends to add homologuous sequences for the Gibson assembly cloning.

Materials:

DNA template, PCR primers, HiFi master mix (Takara), Thermal cycler

Procedure:

Mix together in a PCR tube: 1µl forward primer (10µM), 1µl reverse primer (10µM), 50ng DNA template, 10µl HiFi Master Mix (Takara), Up to 20µl nuclease-free water.

We used the following PCR program : 95°C for 5 minutes (step 1), 95°C for 20 seconds (step 2), 57°C for 10 seconds (step 3), 68°C for 1 minute (step 4), 30 cycles between steps 2 to 4, 68°C for 10 minutes, end at 4°C.

Purification of PCR products on agarose gel

Aim:

To isolate and extract the specific fragments corresponding to the expected size (digested vector and modified inserts).

Materials:

1% agarose gel, 1X TAE buffer, DNA ladder, SyBR safe, Loading buffer, Blue-light transluminator, Nuclease-free water, Gel extraction kit (Stratagene), DNA purification kit (Sigma-Aldrich), Heat block

Procedure:

A 1% agarose gel is prepared with SyBR Safe dye for gel electrophoresis. 2 ul of ladder were loaded on the agarose gel. 3 ul of loading buffer were added in each PCR samples and digested vector and they were loaded on the gel. Migration of samples at 100V for one hour. Visualization under a blue-light transilluminator. The bands corresponding to the expected size of the PCR products were cut and agarose slices were dissolved into a gel extraction buffer (Gel Extraction kit from Stratagene) at 50°C for 10 minutes. After dissolution of agarose slices, DNA is purified with a DNA purification kit by following instructions of the manufacturer (Sigma-Aldrich). DNA is resuspended in Nuclease free water and DNA concentration was measured with a Nanodrop.

Gibson Assembly cloning for the gene construct

Aim:

Cloning of inserts into destination vectors.

Materials:

Digested pHAGE lentiviral vector, Purified insert from previous step, in-fusion HD cloning reagent (Takara), Heat block

Procedure:

50ng of digested pHAGE vector and 100ng of insert were mixed together in a total volume of 8µl. 2µl of the in-fusion HD cloning reagent (Takara) were added and mixed by pipetting up and down. Incubation at 50°C for 15 minutes and then tube is put on ice before transformation.

Transformation of the cloning product

Aim:

To select and amplify cloning products.

Materials:

Stellar competent bacteria (Takara), SOC medium, LB agar-plates + 100µg/ml ampicillin, Cloning product

Procedure:

1µl of the cloning mixture were used to transform 50µl of stellar competent bacteria (Takara), mixed by flicking the tube, and incubated on ice for 30 minutes. Bacteria were heat shocked for 60 seconds at 42°C and incubated on ice. Bacteria were transferred in 500µl of SOC medium and incubated under shaking at 37°C for one hour. Bacteria suspension was spread on ampicillin-containing LB agar plates and incubated overnight at 37°C to select for positive clones.

Screening for positive clones and amplification

Aim:

To identify positive colonies corresponding to insert-containing vectors.

Materials:

PCR primers, GoTaq PCR master mix (Promega), Thermal cycler, DNA ladder, DNA loading dye, 1% agarose gel, 1X TAE buffer, LB liquid culture, Midi-prep kit (Invitrogen)

Procedure:

Colonies are screened by using PCR on colony with one primer specific to the vector and one primer specific of the insert to check both insertion and orientation of the insert inside the pHAGE vector. In a PCR tube, we mixed: 1µl forward primer, 1µl reverse primer, A picked colony, 10µl GoTaq PCR Master Mix, Up to 20µl of nuclease-free water.

The following PCR program was used: 95°C for 5 minutes (step 1), 95°C for 20 seconds (step 2), 60°C for 10 seconds (step 3), 72°C for 1 minute (step 4), 30 cycles between steps 2 to 4, 72°C for 10 minutes, end at 4°C.

PCR products were loaded on a 1% agarose gel after adding 3µl of loading buffer. After migration at 100V and visualization under blue-light transilluminator, positive clones showed a band at the expected size of the insert. A positive colony is picked up and seeded in liquid LB culture medium with ampicillin overnight at 37°C under agitation. DNA is then extracted by using a midi-prep kit (Invitrogen) according to the instructions of the manufacturer. DNA is sequenced to check the quality of the sequence of the construct.

Generation of stable reporter cell lines

Production of lentiviruses

Aims:

To produce lentiviruses that contain reporter constructs to generate stable reporter cell lines.

Materials:

Plasmids: customized pHAGE vectors (containing the reporters), psPAX2, pMD2G. PEI, Red DMEM medium and HEK293T cells

Procedure:

Seed HEK293 cells in 10cm-dishes at 4 million cells/dish. Incubate for 24 hours at 37°C. Cells are transfected with a mix of the plasmids pMD2G, psPAX2 and the lentiviral reporter plasmid of interest by using a PolyEthyleneImine transfection method. Add the transfection solution to the cells, and incubate overnight at 37°C. Perform a complete medium change. Incubate cells at 37°C for 8 hours. Collect the medium, which contains lentiviruses, into a tube. Add complete medium to the cells. Repeat collection and refilling with media twice more at 24 hour intervals.

Lentiviral transduction

Aims:

To generate a stable reporter cell lines by using lentiviruses that stably integrate the reporter construct into the host genome.

Materials:

Red DMEM medium, Collections of lentiviruses-containing supernatants, Puromycin

Procedure:

Cells used to generate the stable reporter cell line are seeded directly into the lentiviruses-containing medium, with the same density as for normal cell culture, at 37°C for 96 hours. Aspirate Lentiviruses-containing medium and replace with complete cell culture medium. Add puromycin at a final concentration of 2μg/mL directly into the culture medium for 24 hours, which selects for antibiotic resistant (reporter+) cells. Remove puromycin culture medium containing dead cells and replace with complete cell culture medium. The stable reporter cell line is maintained in a medium with 1μg/ml of puromycin.

Fluorescent labeling of alginate

Aims:

To make fluorescently-labeled alginate for encapsulation of cells.

Materials:

Fluorophore, Alginate, EDC, Sulfo-NHS, Slide-A-Lyser cassette (10K), MES buffer, DMSO

Procedure:

Staining of alginate with fluorophore bearing an amine group: (1) Prepare 25 mL of 1% (w/v) Alginate solution in 0.1M MES pH 6.0 and mix overnight on a rotator at room temperature (RT). (2) Add 1 mg of the fluorophore that is dissolved in 200μl DMSO and let mix with the alginate for 10-30 minutes RT on a rotator. (3) Add sulfo-NHS dissolved in 200ul 0.1 M MES pH 6.0 for a final concentration of 2mM in the alginate solution and mix for 30 min RT on a rotator. (4) Add EDC dissolved in 200ul 0.1M MES pH 6.0 for a final concentration of 5 mM in the alginate solution and let mix and react for 2 hours on a rotator at room temperature. (5) Dialyse in a Slide-A-Lyser cassette (10K) in 1L of distilled water for 30 minutes. Then, change the water, put the cassette in 5L of distilled water and leave overnight with gentle stirring at 4°C. (6) Collect the stained alginate from the cassette and transfer it into a plastic tube, store it at 4°C.

Before encapsulation, mix the stained alginate with the unstained alginate (prepared by doing step 1 only) with a stained: not stained ratio of 1:10.