Experiments

Circadian Rhythms Disruption (CRD) may increase the risk of Alzheimer's Disease (AD) and trigger an inflammatory response in neurons.
According to omics study, Rose Flavone inhibit the signaling pathway NF-kB/TNF-α by activating the expression of grin2B.

Step 1: Create an AD cell model [cell lab]

1. Treat Cells: Create an AD cell model by treating neuronal cells with specific methods (e.g., stimulating inflammatory factors or mimicking CRD).
2. Measure Inflammatory Factors: Record the levels of inflammatory factors in the cells to establish baseline data for the AD cell model.
3. Observe cellular responses: monitor cellular interactions, such as inflammatory signaling, to assess the accuracy of the model.

Step 2: Rose Flavonoid Treatment [cell lab]

1. Treat Cells: Treat the AD cell model with rose flavonoids.

2. Test new cell model: Measure the level of inflammatory factors in the treated cells and compare with the standard level in AD cell model.

3. Compare baseline levels: verify the mitigating effect of rose flavonoids on cellular damage.

Step 3: omics study [molecule lab & third-party testing company].

1. Sample treatment: treat the cell sample and preparing mRNA samples for omics sequencing and analysis

2. Analysis omics data: use data processing tools to analysis data and identify relevant signaling pathways and genes. Identify the function of proteins and genes.

3. See omics study report： https://2024.igem.wiki/squirrel-beijing/omicsstudyreport

Step 4: Verification of Protein Function

1. Treat cells: treat cells with protein encoded by grin2B.
2. Confirm the gene: Measure the level of inflammatory factors in treated cells and compare with standard level in AD cell model to confirm the protein’s function.

Step 5: Product Design

1. Protein Production: synthesize the protein with E. coli.
2. Products designing: based on the experimental results, develop products containing rose flavone or the protein as an alternative to rose flavonoids.
3. See details in Entrepreneurship: https://2024.igem.wiki/squirrel-beijing/entrepreneurship

1. See our daily records： https://2024.igem.wiki/squirrel-beijing/notebook
2. R*Blue sentences refer to detailed information of each step.
3. RCellular experiments were responsible for simulating nerve cell damage due to circadian rhythm disruption and verifying the protective effects of rose flavonoids at the cellular level. Molecular experiments were then performed to further reveal the signaling pathways activated by rose flavone and the synthesis of specific proteins.

Details of procedure in molecular lab

Conducted to analyze the level of specific proteins to help verify inflammatory level and confirm the effect of different treatement

SDS-PAGE Electrophoresis

The concentration of separating gel is 10%

1. 4X SDS-PAGE separating gel buffer 2.5mL
2. ddH2O 4.1mL
3. 30% acrylamide (29:1) 3.3mL
4. 10% ammonium persulfate 100μL
5. TEMED 10μL

The concentration of stacking gel is 5%

1. 4X SDS-PAGE stacking gel buffer 1.25mL
2. ddH2O 2.84mL
3. 30% acrylamide (29:1) 0.83mL
4. 10% ammonium persulfate 75μL10% ammonium persulfate 100μL
5. TEMED 7.5μL

immunohistochemical reaction and ECL

Detailed procedures

1. Electrophoresis, 22V for 90 minutes
2. protein transfer, 4.5W for 90 minutes
3. Blot with 5% skim milk powder for 2-4 hours
4. Wash with 1× TBST buffer 3 times for 5 minutes each
5. Incubate with primary antibody (1/1000) for 2 hours
6. Wash with 1× TBST buffer three times for 5 minutes each
7. Incubate with secondary antibody (1/3500) for 1 hours
8. Wash with 1× TBST buffer three times for 7 minutes each
9. Add 100 μl super ecl plus for each membrane to chemiluminescence, then
10. automatically exposing in the device

Details of procedure in cell lab

cell media change

During the cultivation process, regularly replace the culture medium to maintain cell growth, and observe cellular morphology under the microscope to ensure cells meet experimental requirements.

Detailed procedures

1. Disinfect and take out the cells from the incubator
2. Examine the survival and growth status of each culture flask
3. Disinfect the culture flasks and put them into the laminar flow cabinet
4. Discard the old medium and rinse with 1× PBS buffer, taking care not to dislodge the cells
5. Add 6 mL of fresh medium
6. Seal the cap, covering the bottle opening with a sealing film
7. Disinfecting culture flasks and put them back to the incubator

cell passaging

By performing cell passaging, increase the number of cells available for the experiment to ensure its smooth progression.

Detailed procedures

1. Disinfect the culture flask of neurons and take it into the benchtop
2. Suck the medium into a centrifuge tube
3. Wash with 1× PBS buffer for three times, in order to remove remaining medium
4. Digest with 100μl 0.5% trypsin for 5 minutes
5. Add serum to stop the digestion, resuspending, and collect the cell suspension into a centrifuge tube
6. Take out the centrifuge tube from the benchtop, and centrifuge at 1500 rpm for 5 minutes
7. Disinfect the tube and return it to the benchtop, aspirating the supernatant and discarding it, adding fresh medium, and resuspending the cells
8. Divide the cell suspension evenly between two culture flasks, toping up the medium to 6 mL in each culture flask, sealing and label each
9. Take the culture flasks out, disinfecting and putting them back to the incubator.