Cell Culture

Experiment 3 Continued:

APOE Transcytosis Assay and Full System Analysis

Purpose: Show APOE has positive affect on transcytosis, show liposome+APOE+EGF gets endocytosed after going through endothelial cell layer, compare liposome+APOE+EGF that has transcytosed to liposome+APOE+EGF that is added to U251 monolayer.

New liposomes are used for this experiment, therefore the problem with small liposomes in last experiments should not be present here.

2 plates:

Plate 1

Cells:

• 9 P19 b.END3 cells seeded on transwells at 510^50.3 cells.

Conditions:

• 100 µM liposomes
• 100 µM liposomes+APOE
• 100 µM liposomes+APOE+EGF

Ran in triplicates.
Protocol will be same as the one used for experiment C, last run.

Plate 2

Cells:

• 6 P19 b.END3 cells seeded on transwells at 510^50.3
• 6 P10 U251 cells seeded below them at 70000 cells
• 4 P10 U251 cells seeded seperately at 70,000 cells

Conditions:

• 100 µM liposomes
• 100 µM liposomes+APOE
• 100 µM liposomes+APOE+EGF

Ran in duplicates for each setting.
Liposomes will be incubated with all settings for 2:30 hr, after this cells will be fixed and looked at with Cellomics.

Day 0:

• When aspirating media after centrifugation, some of the cells were aspirated up
• Final count is 293 live cells in 3 squares-> 1,953,333 cells total
• Adding 13 µL of cells to each well
• Put 600 media in wells, and 100 µL in transwells, aftern an hour added cells
• After adding cells, added another 100 µL on top, to have better mixing (did Ina’s protocol for mixing each 3 wells for each plate)

Day 1:

• Seeding U251 P11 into wells for plate 2
• Counted 229 cells in 5 squares->916000 cells total
• Adding 5.5 media to cells to make total volume 6.5 mLs and concentration of cells ~h 140000 cells/mL
• Putting transwells in adjacent wells, aspirating media out using pump
• Adding 500 µL of cells to each well, adding 100 µL of media to the ones with transwells on them to make total volume those wells 600 µL

Day 2:
Present: Angel, Amir

Calculations for liposomes concentrations:

Protocol is as follows:

1. Bring in 8 15 mL falcon tubes and label them
2. Bring in 3 syringes and 3 filters
3. Dilute liposomes inside media
4. Syringe filter liposomes
5. Dilute PBS/BSA, 12 µL BSA in 12 mL PBS
6. Bring experiment D plate in, put transwells on adjacent wells
7. Take out media from wells very slowly by using P1000, quickly add 600 µL fresh media
8. While holding each transwell with tweezers, aspirate their media and put them back in their well
9. Add 500µL of liposomes to each transwell
10. For wells without transwells, now take out media and add liposomes 2 by 2
11. Mix well and put back in incubator, set time. This needs to be incubated for 2.5 hrs.
12. Bring out transcytosis assay plate
13. Move transwells to adjacent wells
14. Take out media inside wells with pump
15. Add 600 µL PBS+BSA to each well
16. 3 by 3, take out media from inside each transwell by aspirating it out using P1000 while holding it with tweezers and then put the transwell in PBS and add 500 µL of the condition’s liposomes to it
17. Mix well and Put back the plate, take note of time and start timer for 8 minutes
18. Label 9 micro centrifuge tubes for t=8, 9 for t=35, 3 for initial dilutions and 1 for PBS+BSA
19. After 8 minutes, put transwells in adjacent wells
20. Take out PBS+BSA from wells and put in corresponding sample tubes
21. Add 600 µL of PBS+BSA into wells and put transwells back in their wells
22. While it is being incubated, add 250 µL of blanks and initial dilutions to sample tubes
23. At t=35 repeat step 20
24. Discard the plate
25. While experiment D plate is being incubated, conduct flourescence assay on samples
    • Vortex each sample tube for 30 seconds
    • Add 100 µL of each sample to flourescence plate
    • Measure using scan from 495 to 530 with 2 increments
26. At t=2.5 hrs for experiment D plate fix the cells using the following steps:
    • Have a tube with 15mL PBS
    • Add 0.9 µL Hoechst to 6 mL pfa
    • Take out media
      • Take media out with P1000, add to waste beaker
      • Add 200µL PBS to each well (add against wall)
      • Take out PBS
      • Add 400ulPFA with hoechst, time for 7.5 minutes @37C
      • Take out PFA, wash with PBS three times
27. Wrap foil around plate and label and put at 4 C until Cellomics

Time liposomes added to experiment D plate: 7:23
Started adding liposomes to transcytosis plate at 7:41, finished at 7:44, t1= 7:51, t2= 8:19

---

• Hoechst container was empty, had to make DAPI at 9:42, used Sigma batch of 1mg DAPI, added 500 µL of miliQ water to it, mixed and took water out, put in microcentrifuge tube, then added another 500µL and mixed and added to microcentrifuge tube. Vortexed it for 5 seconds.
• Concentration of DAPI is 1 mg/mL, used 3µL in 6mLs to make 0.5 µg/mL DAPI 4% PFA
• Took media out of cells 10:35, started adding pfa at 10:48, put in incubator at 10:53, left in incubator 6 minutes

Results:

• All samples taken from t=8 and t-35 showed blank fluorescence while initial liposomes reading showed flourescence for all liposomes
• We might need higher amount of APOE on liposomes; future steps would be:
  • Characterizing the membrane proteins of cells using western to see if APOE receivers proteins are present
    • If yes then:
      • Redoing the experiment with a higher concentration of liposomes
      • Remaking liposomes with higher amount of APOE
        • If getting good results, adding astrocytes on the abluminal side of transwells and redoing this experiment and the full experiment
    • If not then:
      • Changing cell type to cells that have APOE receivers and repeating experiments