Notebook

[cell lab]

Cell culturing
We received the cells using for experiment on 7.8, then passaged cells at a 1:4 ratio Then, we changed the media each day, and passaged cells every 2-3 days.

Model construction and cell treatment
In the first step, we divided two types of cells–PC12 cells and N2a cells–into four groups, seeded each in one well of a six-well plate. The four groups are:
1. Group M: r modeling group, refers to the cells treated with Aβ protein 42, to represent cell injury
2. Group C: control group, refers to the cells without any treatment
3. Group M+D: refers to the cells treated with both Aβ protein 42 and rose flavonoid
4. Group C+D: refers to the cells only treated with rose flavonoid.
These are down on 7.13
In the second step, after transcriptomics study clarified the function of gene grin2b in the signaling pathway, we also treated cells with the protein it encodes.

[molecular lab]

Western blotting
We’ve done eight times of wb, with 2 days for each (the first day for gel preparation, electrophoresis, protein transferring, and blotting; the second day for incubating with primary and antibody and ECL) The dates of each wb experiment are:
7.12-13
7.14-15
7.17-18
7.19-7.20
8.13-14
8.15-16
8.17-18
8.20-21
The samples using for testing are cells and medium mentioned in “cell lab-cell treatment”. We tested several inflammatory factors of each group, including NF-κB, TNF-α and IL-1β.
The results of ECL are listed in the “result and analysis” part

[preparation room]

Rose flavonoid extraction

7.8 Selected, washed, and dried 100g of fresh rose petals with an oven.
7.9 Weight 1g rose petal powder and extracted the powder with distilled water at a ratio of 1:10 (i.e. 10 mL) in a water bath at 65 ℃ for 2 hours, 3 times.
7.10 centrifuged the extraction solution for 15 min (1500 rpm), then collecting the supernatant–containing all rose flavonoid–together

Western blotting

The concentration of separating gel is 10%
4X SDS-PAGE separating gel buffer    2.5mL
ddH2O                                    4.1mL
30% acrylamide (29:1)    3.3mL
10% ammonium persulfate    100μL
TEMED                                    10μL

The concentration of stacking gel is 5%
4X SDS-PAGE stacking gel buffer    1.25mL
ddH2O                               2.84mL
30% acrylamide (29:1)    0.83mL
10% ammonium persulfate    75μL
TEMED                               7.5μL

The time and other settings
1. Electrophoresis, 120V for 15 minutes, then 140V for 30 minutes
2. Prepared PVDF membranes by one-minute immersion in anhydrous methanol
3. the assembly sequences within the transfer apparatus was organized from the negative to positive pole as follows: sponge, filter paper, gel, membrane, filter paper, and sponge
4. protein transfer under 110V for 90 minutes
5. Blot with 10% skim milk powder for at least 1 hour
6. Wash with 1× TBST buffer 3 times for 5 minutes each
7. Incubate with primary antibody (1/1000) for 2 hours
8. Wash with 1× TBST buffer three times for 5 minutes each
9. Incubate with secondary antibody (goat anti-rabbit IgG-HRP) (1/3500) for 1 hours
10. Wash with 1× TBST buffer three times for 7 minutes each
11. Add 100 μl super ECL plus for each membrane to chemiluminescence, then automatically exposing it in the device

Cell culturing

culturing
Use DMEM medium containing 10% FBS and 1% penicillin/streptomycin
culture in a humidified incubator at 37 ℃ with 5% CO2
cell passage
1. Wash with 1× PBS buffer for three times
2. Digest with 100μl 0.25% trypsin for 5 minutes
3. Centrifuge at 1500 rpm for 5 minutes
4. Divide the cells into two culturing flasks