Cell Culture


Experiment 3:

Analysis of APOE’s Effect on Transcytosis Through b.END3 Cells

Literature Reading:


Source: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4279133/figure/pharmaceutics-06-00557-f001/


Experiment 3 run 1:

Transcytosis assay
Conditions present: liposomes+EGF+APOE, liposomes+EGF+APOE-HZ
Control: Negative-> liposomes
Transwell configuration with PET 0.4µm filters, cells were grown on transwells for two days, initial seeding concentration was 5.0*10^5 cells/mL

Purpose: To see positive effect of APOE on transcytosis.

Procedure:

1. Dilute 4µL BSA into 4mL PBS
2. Dilute Liposomes according to:

  Amount of liposomes µL Amount of Media
Liposome 3.41 996.6
Liposomes+APOE+EGF 7.15 993
Liposomes+APOE-HZ+EGF 51.4 948.6

3. Filter sterilize all liposomes
4. Take out media from bottom
5. Add PBS+BSA into all wells
6. While holding the transwells with tweezers, take out original media inside transwells
7. Add liposomes for the corresponding condition
8. Repeat for all conditions and replicates
9. Incubate for 30 minutes

10. Take out PBS from bottom wells, put into corresponding micro centrifuge tubes and vortex each for 30 seconds
11. Add 300 µL of each of the samples to 96 well plate, add PBS+BSA as blanks
12. Scan for flourescence with SKANit, excite at 495, measure from 505 to 540

Results:

Results show permeability of all wells to the liposomes. In order to ensure intactness of liposomes, DLS was ran on liposomes. All samples showed large variability in liposome sizes. My guess it there is a large amount of small liposomes in each condition that would easily go through the cell layer easily and make large background for the liposomes that actually go through the cells. If this is the case, then those liposomes should be able to go through the cell layer much faster than the larger ones that actually get transcytosed.

As a result, I will run this experiment again, but with a small difference, at t=5 minutes, I will take out the PBS+BSA solution at the bottom of each well and replace it with new PBS+BSA. Those samples at t=5 should include most of the background as the smaller liposomes would go through easily. As for the samples taken at t=30, it will be expected that the liposomes with APOE will have a higher signal. Samples will not be taken at different time points because I don’t want to disturb the experiment too much and I don’t want to dilute the signal too much.
A better future experiment would be to have astrocytes with b.END3 cells to have tighter junctions.

Run 2:

Cells were seeded for this run on October 9th by Amir. The seeding density is 5*10^5/3 cells. There are 8 wells seeded for this experiment. We will have two wells for negative control liposomes, 2 wells for L+APOE+EGF and 2 for L+APOE-HZ+EGF. New liposomes will also be used and they will have 2 wells.

Also 500µL of 100 µM liposomes will be used for this experiment instead of 300µL from last time.

Procedure:

1. Dilute 10µL BSA into 10mL PBS
2. Dilute Liposomes according to:

  Amount of liposomes µL Amount of Media
Liposome-Old 5.12 1495
Liposomes+APOE+EGF 10.73 1490
Liposomes+APOE-HZ+EGF 77.1 1423
Liposomes-New 8.13 1492


3. Filter sterilize all liposomes
4. Using the tweezers, put the transwells in adjacent wells
5. Take out media from bottom wells
6. Add 600µL PBS+BSA into all wells
7. While holding a transwell with tweezers, take out original media inside transwell and then put them in PBS+BSA
8. Add liposomes for the corresponding condition, repeat this for its replicates
9. Repeat steps 7 and 8 for all conditions
10. Incubate for 8 minutes

11. Bring plate into the hood, tap it to the surface 10 times
12. Put transwells in adjacent wells
13. Take out PBS from all wells and put it in corresponding microcentrifuge tubes
14. Add 600µL PBS+BSA to each well and put transwells back into their wells
15. Incubate until t=35 minutes

16. Take out PBS from bottom wells, put into corresponding micro centrifuge tubes and vortex each for 30 seconds
17. Add 300 µL of each of the samples to 96 well plate, add PBS+BSA as blanks
18. Scan for flourescence with SKANit, excite at 495, measure from 505 to 540

Notes:

Samples were put in fridge overnight before analysis.

Expected results:

Experiment 2

Experiment 3 cont.