Team:Jiangsu High School/Notebook
Notebook
Notes: The team has been separated into Group A and Group B, both of which would alternate between Dry and Wet Lab for the following days.
July 17
Both groups met with Mr. Shen—the secondary PI of the team—to discuss the schedule as well as how work would be distributed between the members for the next two weeks. Dr. Lu, the Wet lab director, then introduced the objective of our experiment: testing which compound would incite cell autophagy in the brain to combat AD (Alzheimer's Disease), where the main cause is accumulation of harmful proteins. Dr. Lu also pointed out the social impacts of AD in a world with a large geriatric population. Ms. Wang, the director of Dry lab, then supervised the design of the team uniform and logo.
July 18
Dr. Lu and Dr. Lv, the co-director of the Wet lab, went through the safety protocol in the lab before supervising the plasmid transformation process. The lab transformed normal E. coli into competent cells with TagRPF-mWasabi-LC3 (the plasmid DNA). When cells are placed in a 0 degrees Celsius CaCl2 solution, E. coli bacteria would become more permeable to the plasmid DNA. Then, after the cells are placed on ice along with the DNA, the solution would get heat-shocked at 42 degrees Celsius for about 2 minutes. This would promote the absorbance of the genetic material. The plasmid would then give the bacteria resistance towards a specific type of antibiotics, which could be used in the screening process.
We also started to breed and incubate cells today. The aim of this is to cultivate SH-SY5Y bacteria cultures for later research and experimentation. Even though SH-SY5Y cannot completely imitate body conditions as neural cells could, the experimenters chose the former because the latter is not possible to cultivate at an efficient pace, not possible to choose between specimens, and not suitable for later experiments involving autophagy.
July 19
The group performed the second trial of plasmid transformation so there would be more samples for the following stages. The group also bred more lines of cells in the incubation box.
July 20
The group amplified the plasmid DNA via the shaking incubator, extracted the plasmid DNA from the other proteins that might be present in the solution, and purified the solution in order to isolate the plasmid DNA. This experiment would use alkaline lysis in order to extract plasmid DNA. In the pH range of 12-12.5, the double-helix structured DNA would undergo denaturation. In contrast, the hydrogen bonds within the circular-shaped plasmid DNA would break, but the two complementary strands would still be connected. When added pH 4.8 acid potassium acetate to lower the pH of the solution, the plasmid DNA would undergo renaturation whereas genomic DNA would not because of the bigger size of its molecules. Using the centrifuge, most of the cell debris, genomic DNA/RNA, and protein would become sediments at the bottom of the tube. All of the plasmid DNA would be in the supernatant. Using the cation column, isopropanol to induce sediments, and ethanol to rinse the solution, the experimenters would extract the purified plasmid DNA.
We also developed continuous cell lines and changed the cell medium to give the cells more resources (both nutrients and space) to encourage more replication. We changed the liquid medium used in the petri dishes after moving the cells into a bigger petri dish.
July 21
The group in the lab repeated the cell medium change, development of cell lines, and plasmid amplification of the cells from Day 2.
July 22
The group used lipofection to implant LC3 into the cells. Transfection is a method of implanting foreign genetic material into a cell. There are different methods of transfection: for this experiment, we are using lipofection. Lipofection is association of nucleic acid with a cationic lipid solution. The resulting molecular complexes (called lipoplexes) then enter into the cells’ genomic information. By entering the nucleus, transfection could then replicate its genetic material within its host.
July 23
The group in the lab repeated the same process of lipofection performed by the other group yesterday.
July 24
Today we prepared the solutions to see the effectiveness of each well in response to the autophagy flux test which would happen the 26th. Macroautophagy is a process in which autophagosomes sought to degrade and “recycle” damaged proteins or impaired organelles after fusing with lysosomes to turn into autolysomes. Autophagy is being pushed further into the spotlight in the recent years because how its dysfunction may lead to many neurodegenerative diseases such as Alzheimer’s Disease (AD) (Zhang et all, 2013). Damage in the autophagy mechanism is directly linked to the protein accumulation which would increase toxicity in neuro-pathways of animal models. Mutations in presenilin-1 caused primarily the failure of autophagy to breakdown protein; this would eventually lead to neuronal cell death in AD models (Lee et all, 2010).
We also put the cell samples in frozen storage (-80 degrees Celsius) because they would not be used for a period of time to save space for samples that are more frequently used in the -20 degrees Celsius storage. This procedure is to prepare cell samples for long term frozen storage. Prepare the frozen storage solution (90% liquid culture medium and 10% DMSO). In our case, we are making 3.5mL of storage solution so we will add 3.15mL of liquid culture medium and 0.35mL of DMSO. It is essential to add DMSO into the solution before it lowers the freezing point of the cells as well as prevents the cells from losing moisture and crystalizing (this would destroy the cell structure). After pipetting the liquid medium out of the petri dishes, we will deliver the frozen storage solution and mix it well with the cells on the dish. Making sure to loosen their grip on the dish walls, transfer the solution to three separate tubes (1mL in each).
July 25
We chose Mingjian Zhu as the head captain, Yifei Shan as the head of the Dry lab and Shuwen Zhang as the head of the Wet lab. The group performed a second trial of the preparation for the autophagy flux on the next day. Additionally, the group also put their cell samples in frozen storage.
July 26
Both groups collected data and analyzed the photos we got from the confocal microscope. We will use the photos to determine which compound is most effective at inciting autophagy. This procedure is best done in a dark room with no direct light. Because the fluorescence inside the cells may be damaged from white light. Deliver PBS solution into each well in order to rinse the wells of damaged or dead cell material. Make sure the solution completely submerges the base of the well. Make sure the pipette tip is placed against the well's wall when delivering the solution. Now, add 4% PFA solution in order to fix the cells onto the circular glass slides placed in the wells before the plasmid transfection. Make sure the pipette tip is held against the walls when delivering the solution. The solution should submerged the base of the wells. In a microscope slide container, place down 6 glass slides (since 2 solutions could be placed per slide). Use a tweezer to take out the circular slides from each well. After taking out the slide, be sure to rotate the slide 180 degrees so that the side with the cells would face downwards and be in contact with the anti-fade mounting medium. Take the microscope slide container to the confocal microscope. Take out a slide. Make sure to rinse the lens with 75% ethanol with tissue paper before placing the slide on for examination. Use the knobs to adjust what you can see until you see the clearest and most (in number) cells. The more cells and the better they are stained, the more likely their other fluorescent tags would show. Using the Leica computer app, press "Live" at the bottom left corner to get the view from the microscope and you could use the knobs as well to adjust your field of vision. We have 3 different sequences (the Hoechst blue stain, the mWasabi green fluorescent light, and the red fluorescent light). For each one, pan around the field of vision until you've decided you have the best representation of the compound's autophagy mechanisms. When done, rename the project to your compound name to not confuse it with the other compounds. Repeat this process until you are done with all of the compounds in the microscopic slide box.
July 27
Both groups were present for the fundraiser that took place in the Global Finance Center in Nanjing. See Dry Lab Notes.
Jul. 19-20: Plasmid Transformation & Selection.
Materials:
E. Coli TOP10
Plasmid, with mTapRFP-mWasabi-LC3, and ampicillin-resistance genome.
LB medium in both liquid and solid states
Ampicillin with a concentration of 50mg/ml, solved in ddH2O
Procedure:
1. Medium preparation: LB liquid medium was prepared with the formulation of: 10g of tryptone, 5g of yeast extract, 10g of NaCl, with a moderate amount of NaOH to adjust pH to 7.0, dissolved in 750ml ddH2O so that a final volume of 1L was yielded. LB solid medium was prepared by a mixture of 200ml of liquid medium with 4g of agar powder. Both medium was sterilized under 120˚C. Once solid medium cooled down under 55˚C, it was added with 10mg of ampicillin, and was immediately plated onto several Petri dishes.
2. 2μl of DNA plasmid was added and mixed into 50μl of competent bacteria (E.coli), in EP tubes, and was placed in ice for 30min.
3. EP tubes filled with bacteria and plasmid was bathed under 37˚C water for 42s, and was then immediately placed back in ice for 3 mins.
4. 1ml of liquid medium was added into tube. The competent bacteria was recovered under 30˚C, 150rpm for 45min.
5. 300μl of bacteria suspension was applied evenly on solid medium. After 15mins of standing, Petri dishes was inverted to incubate bacteria overnight at 37˚C.
6. After overnight incubation, a single bacteria colony was selected to ensure the consistency of genetic factor. Using pipette tip, the colony was picked and put in centrifugal tube, filled with liquid Petri medium, for a 2nd overnight incubation at 37˚C, 150rpm.
Jul.20: Neuronal Cell Recovery
Materials:
SH-SY5Y (bought from ATCC cell bank)
DMEM/F-12 medium
Procedure:
1. Neuronal cell was taken from -80˚C fridge and was bathed in 37˚C water for 2 mins, or until completely melted. DMEM/F-12 medium was taken from 4˚C fridge and was bathed in 37˚C water for 10 mins.
2. 15ml of cell suspension was added to centrifuge tube, along with 4ml medium. It was centrifuged at 2000rpm, 5min, to remove cryoprotectant DMSO.
3. The supernatant was removed, and 5ml medium was added. Blow it gently with pipette.
4. The cell suspension was moved into Petri dish, which was then placed in the incubator.
Jul. 21: Plasmid Extraction and Purification
Materials:
E.coli TOP10, cultured overnight
TIANpure Midi Plasmid Kit II (Cat.no. DP107)
Containing:
· Buffer P1, P2, P3, PD, PW, TB
· Filtration column CS
· Spin Column CP4
· Collection tubes 2ml
Procedure:
1. Centrifuge tube, filled with bacteria suspension, was balanced and centrifuged at 4000 rpm, 15min. The supernatant was removed.
2. 500 μl buffer P1 was added into the centrifuge tube. The bacteria was thoroughly re-suspended using vortex mixer. The suspension was moved into a small tube.
3. 500 μl buffer P2 was added and mixed thoroughly by inverting the tube 6-8 times.
4. 700 μl buffer P3 was added, the tube was inverted 6-8 times for mixing. Cloudy white precipitate was observed. Centrifuge for 12000 rpm, 10 min, and a compact white pellet formed at the bottom of the tube.
5. The supernatant was transferred into filtration column CS (placed in a collection tube). Centrifuge at 12000 rpm, 2 min.
6. Spin column equilibration: The spin column was placed in a collection tube. 500 μl buffer BL was added to CP4. Centrifuge at 12000 rpm, 1 min. The flow-through was discarded, and CP4 was set into a new collection tube.
7. The flow-through in step 5 was transferred to spin column CP4. Centrifuge at 12000 rpm, 1 min. The flow through was discarded.
8. Protein residue was washed away by adding 500 μl buffer PD, and centrifuging at 12000 rpm, 1 min. The flow-through was discarded.
9. The spin column was rinsed by adding 600 μl buffer PW, and centrifuging at 12000 rpm, 1 min. The flow-through was discarded.
10. Repeat step 9.
11. CP4 was placed in room temperature with lid open for several minutes to vaporize residual ethanol from wash buffer PW.
12. Centrifuge at 12000 rpm, 2 min to further remove buffer PW residue.
13. CP4 was placed in a new centrifuge tube, DNA plasmid was eluted by adding 40 μl buffer TB to the center of spin column, and centrifuging at 12000 rpm, 1 min.
14. The concentration of plasmid was tested as the absorbance of the solution. The concentration fell within the range of 800-1100ng/μl.
Jul. 22: Neuronal Cell Passage and Plating
Materials:
DMEM/F-12 complete medium (with serum and penicillin-streptomycin)
Phosphate Buffer Saline (PBS)
Neuronal cells from previous procedures
Procedure:
1. Neuronal cells was taken from the incubator and observed under microscope to make sure they adhere to the wall of the Petri dish.
2. Old culture medium was removed and the Petri dish was rinsed with 4ml of PBS.
3. 1ml of pancreatic enzyme (0.25% concentration) was added, and removed to digest the pseudopodia of neuronal cells.
4. After 1 minute of digestion, 4ml of complete medium was added to terminate digestion. The neuronal cells were detached using pipette.
5. Take 1 large Petri dish, 9ml of culture medium and 1ml of cell suspension from step 4 was added. After mixing, it was labeled and placed in the incubator for passage.
6. Take 1 culture plate with holes. A circular coverslip was put into each hole. Add 1Every hole was then filled with 1ml of cell suspension from step 4. The plate was then labeled and placed in the incubator.
Jul. 23: Plasmid Transfection
Materials:
Lipofectamine(LIP) 2000
Plasmids and Neuronal cells from previous procedures
OMEM minimal medium
MEM minimal medium
DMEM/F-12 complete medium (with serum and penicillin-streptomycin)
Procedure:
1. 2 EP tubes was taken. 600μl of OMEM was added into each tube respectively.
2. 12 μl LIP2000 was added into one of the tubes, and was mixed using pipette. Let stand for 5 min.
3. 6μl plasmid suspension was added into the other EP tube, and was mixed gently using pipette.
4. The solution that contained plasmid was transferred and gently mixed into the tube filled with LIP2000. Let stand for 25 min.
5. Take out the culture plate with neuronal cells. Old culture medium was removed, and 800μl MEM minimal medium was added into each hole.
6. 200 μl plasmid-LIP2000 solution was added into each hole, and a final volume of 1ml was yielded for each hole. The plate was again placed in the incubator for 6h.
7. After 6h, the plate was taken out. All old culture media were removed, and 1ml DMEM/F-12 was added into each hole.
Jul. 24: Compound Addition
Materials:
Various compounds selected as autophagy activator
Neuronal cells from previous procedures
DMEM/F-12 complete medium
Procedure:
1. The culture plate with neuronal cells was taken out of incubator. All old culture media were removed.
2. 12 EP tubes was taken 1ml DMEM/F-12 was filled into each tube.
3. The compound was added into each tube with the appropriate amount that resulted in a final concentration of 1μmol/ml.
4. Each solution was mixed with pipette, and was transferred into each hole of the culture plate.
Jul. 25: Microscope Slide Preparation
Materials:
Neuronal cells from previous procedures
Phosphate Buffer Saline (PBS)
Paraformaldehyde fixation solution (4% concentration)
Hoechst stain
Antifade mounting medium
Procedure:
1. Take out the culture plate with neuronal cells, all old media were removed.
2. Rinse every hole 3 times with PBS.
3. 200μl paraformaldehyde was added. Let stand for 25 mins.
4. The solution was removed and each hole was rinsed with PBS.
5. (All procedure from this on were performed under dark environment) 200μl Hoechst stain was added to give color to cell nucleus. Let stand for 10 mins.
6. The solution was removed and each hole was rinsed with PBS.
7. Take out microscope slide. Antifade mounting medium was applied to the slides. Each coverslip in the hole was carefully taken out, and was inverted to put on the glass slide. The slide was marked with compound name, and was stored under dark environment.
Jul. 26: Autophagic Flux Monitoring
Materials:
Neuronal cell slides from previous procedures
Procedure:
The neuronal cell slide was put under Leica confocal microscope to monitor autohagic flux.