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



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

Plate 2



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:

Day 1:

Day 2:
Present: Angel, Amir

Calculations for liposomes concentrations:

Exp 1 Liposome Calculations Numbers from Nathan
Concentration of Liposome + APOE + EGF (µM phospholipid)   7.49E+03
Concentration of Liposomes + APOE 7.49E+03
Concentration of Liposomes (µM phospholipid) 1.85E+04
For Liposomes + APOE + EGF  
Concentration aims(µM phospholipid) 100
Number of wells for each condition 10
Volumes of each concentration needed (µL) 4500
Dilution needed (µL) 60.0
Volume of media added 4440
Concentration of Liposomes + APOE 7.49E+03
Concentration of Liposomes (µM phospholipid) 100
Number of wells for each condition 5
Volumes of each concentration needed (µL) 3500
Dilution needed (µL) 46.7
Volume of media added 3453
Concentration of Liposomes (µM phospholipid) 2.94E+04
Concentration of Liposomes (µM phospholipid) 100
Number of wells for each condition 7
Volumes of each concentration needed (µL) 4500
Dilution needed (µL) 15.3
Volume of media added 4485

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

Wells for fluorescence assay


Experiment 3

DLS Sizing