{% extends "layout.html" %} {% block title %}Experiments{% endblock %} {% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %} {% block page_content %}
1. 0.1M PBS 2. 0.2M EDC·HCl 3. 0.2M NHS 4. 10kDa Ultrafiltration tube (Catalog number: UFC5010BK) 5. Stock Nanoparticle solution 6. Antibody solution
1) Transfer 200 μL of our bio-synthetic nanoparticles and 0.4mg (8μL in volume) chemo-synthetic
nanoparticles
to two eppendorf tubes.
2) Add 200 μL 0.1M PBS to the second tube to make up for the difference
3)Then add 200 μL 0.1M PBS to each of the tubes to mix it thoroughly.
4)Then, add 60 µL of 0.2 M EDC·HCl and 120 µL 0.2 M NHS to each tube.
5)After 10 min, add 30 μg antibody to the EDC/NHS-activated NPs solutions.
6)After 24 h, transfer the solution to 10 KD ultrafiltration tubes and centrifuge for 20 min
at 14000 rpm to remove the unbounded antibody.
7)Finally, collect the obtained antibody-conjugated NPs (IONPs-antibody) in EP tubes.
1. DMEM High Glucose H-21 Media (Catalog number: 11965084)
2. RPMI 1640 basal Media (Catalog number: 12633012)
3. Fetal Bovine Serum (Catalog number:10099141C)
4. Penicillin, Streptomycin
5. Trypsin/EDTA
6. Dulbecco's Phosphate Buffered Saline
7. Cell strains
8. Tissue culture dish
9. 15 ml Falcon tube
10. 37ºC water bath
11. 37ºC 5% CO2 incubator
12. Insulin (Catalog number: I860440-25mg)
Culture medium: 450 ml DMEM High Glucose H-21 Media, 50 ml (to 10%) Fetal Bovine Serum (Catalog number:10099141C) , 5 ml Pen Strep
Thawing Cells:
1. Take one vial immediately in 37ºC water bath. Transfer the content to a 15 ml Falcon tube already
containing 10 ml of fresh medium.
2. Spin down at 1200 rpm for 3 min at 4ºC. Aspirate supernatant and resuspend cells in fresh medium
and transfer to 10cm tissue culture dish and cultured at 37°C in 5% CO2. Change medium about
every 3 days.
Subculture
After cell culture reaches 80-85% confluence, subculture is conducted.
1. Remove the medium from the dish.
2. Wash 1x with 10 ml of Dulbecco's Phosphate Buffered Saline (without Ca2+ or
Mg2+)
3. Add 1 ml of 0.05% Trypsin and trypsinize for 3-5 min at 37°C.
4. Add 8 ml of medium to neutralize the trypsin and use it to rinse the dish by pipetting up and down
4-5 times to detach the cells off.
5. Spin down at 1200rpm for 3 min. Aspirate supernatant.
6. Add 8 ml of medium to the 15ml tube with cell pellet, and pipette up and down to mix. Subculture
ratio is about 1:10.
7. Put the cells into 37°C with 5% CO2.
Culture medium: 500 ml RPMI 1640 basal Media, 100 ml (to about 20%) Fetal Bovine Serum, 5 ml Penicillin, Streptomycin, 2μg/ml insulin
Thawing Cells:
1. Take one vial immediately in 37℃ water bath. Transfer the content to a 15 ml Falcon tube already
containing 8 ml of fresh medium.
2. Spin down at 1000 rpm for 3 min. Aspirate supernatant and resuspend cells in fresh medium and
transfer
to 10cm tissue culture dish and culture at 37°C in 5% CO2. Change medium about every 2 days.
Subculture
After cell culture reaches 70-75% confluence, subculture is conducted.
1. Remove media from dish.
2. Wash 1x with 10 ml of Dulbecco's Phosphate Buffered Saline.
3. Add 1 ml of trypsin/EDTA for about 10 min at 37°C.
4. Add 6 ml of medium to neutralize the trypsin and use it to rinse the dish by pipetting up and down
4-5
times to detach the cells off.
5. Spin down at 1200rpm for 3 min. Aspirate supernatant.
6. Add 6 ml of media to the 15ml tube with cell pellet, and pipette up and down to mix. Subculture
ratio
is about 1:3/1:4.
7. Put the cells into 37°C with 5% CO2.
1. Cell Counting Kit-8 (CCK-8) (Catalog number: ab228554, Abcam)
2. 96-well plate
3. 37ºC, 5% CO2 incubator
4. 37ºC water bath
5. 96-well plate reader
6. WST-8 Solution
1. Plate 10,000 cells in 100 µl per well for a 96 well plate
2. Add test compounds into cells and incubate for 24 hours in a 37ºC, 5% CO2
incubator.
3. For blank wells (medium without cells), add the same amount of test compounds.
4. Add 10 µl/well (96 well plate) or 5 µl/well (384-well plate) of WST-8 Solution to each well.
Protect from the light and incubate for 1-4 hours at 37ºC.
5. Measure the absorbance increasement at 460 nm.
1. SDS-PAGE gel
2. PVDF membrane
3. BSA blocking solution
4. TBS-T solution
5. Primary antibody
6. Secondary antibody
7. Exposing reagent
8. Electrophoresis system
9. Power supply
10. Protein ladder