Protocols

Cell Passaging
Usage of the hood:
Turn on the vacuum wait until the pressure goes all the way down turn on the light open until
the mark.
Put the media, tripsin and pbs to the water bath for app 30 minutes.
Inside the hood:
 Take out the media, tripsin and pbs from the water bath and dry them with paper towel.
Then spray ethanol on them before putting them inside the hood.
 Take the cell culture and put it in the hood.
 Take 3 pipettes and new T-flask and put it inside the hood.
 Discard the media.
 Wash the flask once with 5 ml of PBS.
 Discard the PBS.
 Add 1 ml of Tripsin (helps detach the cells from the surface) and make sure that it
spreads all over the surface.
 Incubate for 5 minutes at 37 in the incubator.
 Label the new T-flask.
 Take the cells out of the incubator and check if they are detached. If not, tap on the
sides.
 Add 3 ml of medium to deactivate the tripsin. (1 ml tripsin/ 2ml medium)
 Take out the tripsin and the cells and put them into a 15 ml tube.
 Spin the cells for 5 min, 4 degrees and 1000 rpm. And put another tube with the same
volume across to balance it.
 Remove the supernatant solution from the tube. Be careful not to accidentally remove
the cells.
 Add 1 ml of the new media and mix it by pipetting in/out gently.
 Add 10 ml of the new media to the new T-flask.
 Transfer 50 microliter of cells into the T-flask.
 Put the new cell culture back into the incubator.
>> Chance the medium approximately every 4 days. Passage the cells 1 every week.
Cell Passaging for Macrophages (no Tripsin)
Usage of the hood:
Turn on the vacuum wait until the pressure goes all the way down turn on the light open until
the mark.
Put the media and pbs to the water bath for app 30 minutes.
Inside the hood:
1. Take out the media and pbs from the water bath and dry them with paper towel. Then spray
ethanol on them before putting them inside the hood.
2. Take the cell culture and put it in the hood.
3. Take 3 pipettes and new T-flask and put it inside the hood.
4. Discard the media.
5. Wash the flask once with 5 ml of PBS.
6. Discard the PBS.
7. Add 2 ml of media to the T-flask
8. Use cell scrapers to scrape out the cells in the bottom of the T-flask. Make sure to scrape the
cells unidirectionally. Pipet in and out to make sure you get all the cells in the bottom of the
flask
9. Take the cells from the T-flask and put it into a 15 ml tube.
10. Dilute the cells according to the number of T-flasks. add 2 ml of media. (at least make it a 3
flasks).
11. Label the new T-flasks.
12. Add 9 ml of the new media to the new T-flask (make sure to make it up to 10 ml in total in the
flask).
13. Transfer 50 microliter of cells into the T-flask.
14. Put the new cell culture back into the incubator.
>> Chance the medium approximately every day. Passage the cells every 2-3 days.
Cell Freezing
• Take out the media and pbs from the water bath and dry them with paper towel. Then spray
ethanol on them before putting them inside the hood.
1. Take the cells out of the incubator and in the hood, vacuum out the medium.
2. Wash the cells with 5 ml PBS.
3. Put 5 ml of media into a separate 15 ml tube.
4. Add 10 % (500 ul) of DSMO to the media and label “GP, DMSO&media”.
5. Add 1 ml of media inside the T-flask and scrape the cells with cell scraper. Make sure to scrape
the cells in one direction.
6. Spin the cells at 5 min, 4 degrees and 1000 rpm.
7. Vacuum out the supernatant solution and resuspend the cells with DMSO&media
8. Add the cells to cryovials.
Electrospinning Procedure:
1. Let the cover slips sit in the IPA (polylysine solution) for about an hour.
2. Wait for them to air dry.
3. Place aluminum foil on top of the white Styrofoam.
4. Put the gel solution in the needle.
a. Use a flat needle.
b. Take 1 ml of solution into the syringe.
c. Make sure there are no air bubbles by tapping the syringe.
5. Place the syringe in the black holder.
6. Place the positive cord (pink one) on the tip of the syringe. Place the ground (red cord) on the
white Styrofoam.
7. Place 4 paper rings or cover slips on top of the aluminum foil.
8. Turn on the pump to 10 microliters per minute.
9. Set the voltage to around 15 Kv
10. Attach the red cord to the Styrofoam.
11. Turn on the pumper Turn on the power supply set the timer to 2 minutes (sometimes 3
minutes).
12. After 2 minutes turn of the voltage and the timer.
13. Collect the paper rings or the cover slips with tweezer.
For
Liver:11 Kv PCL-14 Kv
Bone: 17 Kv Brain+Lung: 9 kV
Fat: 11 Kv
Electrospinning of Brain Solution
1. Cut the needle using pliers and sand it so that it has a blunt tip.
2. Transfer brain solution to 3ml standard needle.
3. Coat the paper rings or cover slips in IPA (0.01% poly-d-lysine) for almost an hour.
4. Let the cover slips and papers air dry.
5. The setup is shown below:
The distance between the syringe tip and the ventilation hood must be 11 cm.
6. Arrange the flow rate to be 10 microliters per min.
7. Make sure that there is a continuous air flow.
8. Arrange the voltage to be 10 Kv.
9. Turn on the power supply.
10. Let the samples wait for 5 minutes and 30 seconds (approximately)
11. Transfer the samples to 6-wells.
Immunofluorescence Staining
1. Vacuum out the medium.

2. Wash the wells with 2X PBS. Make sure that the pbs covers all the surface. Tilt the dishes
back/forth to make sure that PBS covers the whole surface.
a. Add 1 ml of PBS to each well.
b. Wait for 5 minutes
c. Vacuum out the medium.
3. Take the 4% PFA (from the fridge) and add 1 ml of PFA to each well. Incubate for 10 minutes.
4. Vacuum the PFA solution out and wash 2X with PBS.
5. Add 200 microliter of 1% Triten solution to each of the wells. Make sure that Triten covers the
whole scaffold surface. Then incubate for 10 minutes.
6. Vacuum out the Triten solution.
7. Wash 2X with PBS.
8. Add 200 microliter 5% BSA in each well.Incubate for 30 minutes in the moist environment.
a. How to create the moist environment
i. Get the big white Styrofoam box with cap.
ii. Moisturize the paper towels with water and place them in the box.
iii. Put the plates inside the box and close the cap.
9. Wash each well twice with PBS.
10. Get the antibody. Dilute the antibody with PBS. The dilution ratio that Deep uses is
200(PBS):1(antibody) (look online for dilution ratios). Deep adds 1 microliter of antibody for
every 200 microliter of PBS. Pipet in/out 10-25 time to make sure the solution is mixed properly.
11. Put 1 micron of diluted solution into each well.
12. Store the wells in the white container. Keep the container moist by putting paper towels around
it.
13. Put everything back into the fridge. Incubate overnight. (for looking at small proteins)
a. Or let it sit for 1 hours in PBS.
14. Dilute the secondary antibody with PBS. (look up the ratios on your own but Deep uses 1 micron
(antibody):300 micron (PBS))
15. Add the diluted secondary antibody to each well. Incubate for 1 hour.
16. Wash 2X with PBS. Cover the well to avoid sunlight.
17. Add the staining material (Dappy) once you are in the imaging room. Do not add more than a
drop.
18. Turn on the electron microscope.
19. Turn on the device next to the microscope.
20. Press the green botton under the microscope.

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Cell Passaging

Usage of the hood:

Inside the hood:

Usage of the hood:

Inside the hood:

1. Vacuum out the medium.

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