Experiments

Cell Maintenance

Cell maintenance is crucial for ensuring the health, viability, and consistent morphology of cells in experiments. For HEPG2 cells, which are human liver carcinoma cells, proper maintenance ensures that they retain their characteristic metabolic functions. Regular monitoring and maintenance of the cells ensures that they are free from contamination, which can compromise the experiment, and instead ensures reproducibility of experimental data, allowing for reliable comparisons.

Cell Splitting

Cell splitting, or subculturing, is a vital aspect of cell maintenance, especially for adherent cells like HEPG2. Over time, cells can become overconfluent, leading to changes in their behavior, morphology, and metabolic activity. Splitting cells prevents overcrowding, ensuring they have adequate space and nutrients to grow and function optimally. It also allows for the removal of dead or dying cells, ensuring a healthy culture.

Preparations

1. Turn on the fan and light of the biosafety hood and open.
2. Spray your gloves with ethanol and allow them to air dry them.
3. Spray the surface of the hood with ethanol and wipe it down with a paper towel.
4. Get pre-aliquoted trypsin (1.5 mL) from the freezer and place under the hood.
5. Get a waste beaker and spray with alcohol; enough so that there is still some pooled at the bottom, and place under the hood.
6. Place EMEM media from the fridge under the hood.
7. Take 2 new T-25 flasks and place them under the hood. Label them with the passage number, the cell type & media type, the split ratio (1:8 or 1:80), the date, & initials.
8. Grab 2 sleeved 10mL pipettes and place under the hood.
9. Put T-25 flask with 70% confluent sample under the hood.

Procedure

1. Loosen caps so they can be easily removed and ensure T-flasks are facing the correct direction.
• If trypsin is still frozen, thaw between hands before continuing.
2. Un-sleeve a 10mL pipette.
3. Take 10mL of EMEM and dispense 5mL into the 1:80 flask and 5mL into the 1:8 flask.
4. With the same pipette, take 6.5mL of EMEM and spray into the 1:8 flask.
5. Use the same pipette to remove the old media from the flask with the sample and discard it into the waste beaker.
6. Take all the pre-aliquoted trypsin and dispense into the flask with the sample.
7. Close the cap for trypsinized cells tightly.
• Incubate the cells with the trypsin for 5-10 minutes to allow them to split.
• Check the cells periodically under the microscope to make sure they are ready for the next step. They should no longer be adhered to the flask and should be floating as just a few cells, not big clumps of cells.
8. Once ready, return the cells under the hood and un-sleeve the second 10mL pipette.
9. Take the extra 6.5mL media from the 1:8 flask and dispense into the cell flask.
10. Suction up the cells, trypsin, and media from the flask, and dispense a few times to disperse any remaining chunks of cells.
11. Take 1mL of this mixture and disperse into the 1:8 flask.
12. Suction and dispense like the previous step to mix.
13. Take .5mL from the 1:8 flask and then dispense it into the 1:80 flask.
14. Suction and dispense to evenly mix.
15. Put flasks in the incubator and clean up.

DNA Isolation

DNA extraction involves utilizing various reagents and centrifugation to isolate pure DNA. A specialized DNA extraction kit, equipped with the necessary washes and buffers, can be employed for this purpose. For our experiments, DNA isolation is essential since both GFP DNA, and V2 DNA are regularly used. Bacteria that carry these genes are cultivated and incubated, and the extracted DNA is then introduced to cells via transfection.

Day 1: Petri Dish Setup

1. Get bacterial cells from the freezer.
2. Label an appropriate petri dish with cells (V2, GFP, etc.), date, and initials.
3. In the sterile area of a Bunsen burner, use a sterilized inoculation loop to streak the petri dish with bacterial cells.
4. Incubate at 37 °C for 24 hours.

Day 2: Bacterial Transfer

1. Obtain a petri dish from the incubator.
2. Prepare a 250mL baffled Erlenmeyer flask of 50mL of LB growth medium and label it with cells (V2, GFP, etc.), antibiotics, date, and initials.
3. Add 50uL of appropriate antibiotic to the flask.
4. In the sterile area of a Bunsen burner, use a sterilized inoculation loop to add bacterial cells to the flask.
5. Incubate the baffled Erlenmeyer flask in a shaker at 37 °C and 300 RPM for 19-20 hours.

Day 3: Isolation

1. Pour liquid from the Erlenmeyer flask into two 50mL tubes.
2. Centrifuge at 3000 RPM for 10 minutes.
3. Pour out liquid and upturn tubes onto a paper towel for 1 minute.
4. Resuspend each cell pellet with 600 µL of P1 Buffer.
5. Label 3 microcentrifuge tubes with cells (V2, GFP, etc.), date, and initials.
6. Split mixture into three microcentrifuge tubes (200 µL each).
7. Add 200 µL of P2 Buffer into the microcentrifuge tube.
8. Add 400 µL of P3 Buffer into the microcentrifuge tube.
9. Mix by inversion until all the pink color has disappeared.
10. Centrifuge 15,000 x g for 5 minutes (more if needed).
11. Remove supernatant (clear yellow liquid) from the microcentrifuge tube without disturbing the cell pellet and put it into a cartridge with a collection tube.
12. Centrifuge 15,000 x g for 1 minute.
13. Dump excess liquid from the collection tube.
14. Add 200 µL of DNA wash to the cartridge.
15. Centrifuge 15,000 x g for 30 seconds.
16. Dump excess liquid from the collection tube.
17. Repeat steps 14-16.
18. Add 200 µL of endo-wash to the cartridge.
19. Centrifuge 15,000 x g for 30 seconds.
20. Dump excess liquid from the collection tube.
21. Repeat 18-20 3X, I.E., 600ul total.
22. Dry centrifuge at 15,000 x g for 30 seconds without added wash.
23. Warm 150 µL DNA elution to 65 C.
24. Add 33 µL of DNA elution to the cartridge and place cartridge inside a microcentrifuge tube labeled with DNA, date, initials, and A (for first dilution).
25. Centrifuge at 15,000 x g for 2 minutes.
26. Repeat steps 24-27 two more times labeling "B" for the second dilution and "C" for the third dilution.
27. Measure each DNA absorbance with a nano dropper.
28. Label tubes with absorbance and ng/µL.
29. Freeze in the cell lab.

Transfection

Transfection is a technique used to introduce foreign DNA or RNA into cells. This technique allows for the manipulation of cellular genetics, leading to the expression or inhibition of specific proteins or functions. In the context of this project, both GFP and V2 are transfected into cells. GFP serves as a control in the luciferase assay due to its ability to emit green fluorescence when viewed under a fluorescent microscope, and V2 is the experimental vector transfected into DNA to be evaluated in the luciferase assay.

Day 1: 96-Well Plate Set Up

1. Remove old media from the pre-setup flask that is about 70% confluent.
2. Add 1.5mL trypsin to the flask.
3. Incubate for about 5-10 minutes, or until the cells are split.
4. Add 6.5mL fresh media to neutralize the trypsin.
5. Resuspend until it is a single cell suspension.
6. Count cells using a hemocytometer.
7. Add 10µL of cells from the flask to hemocytometer.
8. Count the four corner squares.
9. Average the counts of the four corner squares and multiply by 10,000.
10. Get cell stock volume by calculating:
    (Avg Cell Count x 10,000) cells/mL = 30,000 cells
11. Prepare 96-well plate:

Label with HEPG2, the date, and initials. Add media and cell stock according to their count so that: µL cell stock + µL media = 100µL

Incubate 96-well plate to allow cells to attach to the plate and grow. (24-48 hours)

Day 2: Transfection

1. Prepare two microcentrifuge tubes.
2. Label one tube with D to use for the DNA solution.
3. Label one tube with L to use for the Lipofectamine 2000 solution.
4. Add Xµg* (calculated using ng/mL labeled on DNA) of DNA to 25uL of Opti-MEM media into the D tube.
5. Mix DNA solution by inversion three times.
6. Vortex Lipofectamine 2000 for 30sec.
7. Add YµL* of Lipofectamine 2000 to 25uL of Opti-MEM in the L tube.
8. Vortex solution of tube L for 30 sec.
9. Let solutions in both tubes incubate for 5 minutes at room temperature.
10. Remove old media from well in the prepared 96-well plate with 70% HEPG2 cell confluency.
11. Add 100uL of fresh MEM media to the well.
12. Add 50µL of the total solution of DNA and Lipofectamine 2000 to the well.
13. Pipette the total well volume up and down one time to mix.
14. Place the 96-well plate in the incubator for 24 hours.

* X & Y are placeholders until the transfection optimization experiment is completed.

Transfection Optimization Experiment

A transfection optimization experiment is a systematic approach to improve the efficiency of introducing foreign DNA into cells. By testing different concentrations of DNA and reagents, the optimal conditions for maximum transfection efficiency can be identified. Low transfection efficiency can hinder the success of experiments, leading to inconclusive results and wasted resources. By optimizing the transfection process, it ensures a higher proportion of cells receive genetic material.

1. Prepare 10 microcentrifuge tubes.
1. Label five tubes G1, G2, G3, G4, and G5 for the GFP DNA solution.
2. Label five tubes L1, L2, L3, L4, and L5 for the Lipofectamine 2000 solution.
2. Add 25uL x 5 (number of rows) of Opti-MEM to each of the tubes labeled G1 – G5.
3. Add 25ul x 5 (number of columns) of Opti-MEM to each of the tubes labeled L1 – L5.
4. Distribute GFP DNA to the (G) tubes in the following order:

1. 0.5uL to G1
2. 0.9uL to G2
3. 1.8uL to G3
4. 3.6uL to G4
5. 7.2uL to G5

5. Mix DNA solutions by inversion three times.
6. Vortex Lipofectamine 2000 for 30 seconds.
7. Add Lipofectamine 2000 to the (L) tubes in the following order:

1. 0.25ul to L1
2. 0.5uL to L2
3. 1.0 to L3
4. 2.0 to L4
5. 3.0 to L5

8. Vortex L-tubes for 30 seconds.
9. Let both solutions incubate for 5 minutes at room temperature.
10. While the solutions incubate, label 25 more microcentrifuge tubes for their corresponding plate location.

1. For row A, label five tubes A1 – A5.
2. For row B, label five tubes B1 – B5.
3. For row C, label five tubes C1 – C5.
4. For row D, label five tubes D1 – D5.
5. For row E, label five tubes E1 – E5.

11. Once ready, distribute the solutions of tubes G1 – G5 to their corresponding columns:

1. For G1, transfer 25uL to A1, 25uL to B1, etc.
2. For G2, transfer 25uL to A2, 25uL to B2, etc.
3. Continue this process for the rest of the remaining GFP DNA solutions and their columns.

12. Distribute the solutions of the tubes L1 – L5 in the corresponding rows:

1. For L1, transfer 25uL to A1, 25uL to A2, etc.
2. For L2, transfer 25uL to B1, 25uL to B2, etc.
3. Continue this process for the rest of the remaining Lipofectamine 2000 solutions and their rows.

13. Let the 25 solutions of DNA and Lipofectamine 2000 incubate for 15 minutes. While waiting, continue to the next step.
14. Remove old media from the wells in the prepared 96-well plate.
15. Add 100uL of fresh MEM media to each well.
16. Once incubation is complete, add 50uL of the mixed solution to its corresponding well.
17. Pipette the total volume of the well up and down once to mix.
18. Place the 96-well plate in the incubator for 24 hours.
19. After 24 hours, replace the media and document fluorescence with a microscope. Let incubate for 24 hours.
20. After 48 hours total, change the media again and document fluorescence with a microscope. Return to the incubator.

Dual Luciferase Assay

The purpose of this assay is to test whether the V2 circuit detects cholesterol. The luciferase is a reporter gene that determines if the promoter is on or off. The luciferase gene is located inside the circuit, so when the substrate is added to the cells, it creates a reaction. This reaction produces luminescence, which is measured. The luminescence begins to decay after 12 minutes, so it is important to get luminescence readings before then. Low transfection efficiency will affect the results of the assay, so it is important to have multiple control wells to determine what or if anything went wrong in the assay. The ratio of the firefly to Renilla luminescence is compared between the control and transfected wells to determine if the V2 circuit and the assay are successful. Values of luminescence for firefly of the transfected cells should expect to be >10,000.

Must be conducted a minimum of 48 hours after the previous transfection step

Make sure reagents are not older than 6 months.