Cell Culture

Experiment 2:

Fluorescence Permeability Assays

September 23, 2016
Present: Ina

Task: Create a calibration curve for liposome fluorescence

1. Prepared dilutions of liposomes in PBS:

Initial Concentration (µM) 2.94E+04      
Desired Concentration (µM) 0.1 1 10 100
Final Volume (µL) 1000 1000 1000 1100
V1 100.00 100.00 100.00 3.75
Volume needed to add for 2 replicates 900.00 900.00 900.00 1096.25

2. Liposomes seemed like they aggregated, and the solution they are in did not look good. It was very clumpy even after gently tapping the vial to mix the liposomes. When pipetting out the liposomes and diluting them in PBS, aggregates were visible.

3. Transferred duplicate samples of 450 µL each into a clear, round ubottom 96-well plate (no flat bottom ones were available). The plate was read using the SkanIt Varioskan Flash microplate reader, with the Ex/Em set at 495nm/507nm at 300 ms per reading. Fluorescence was taken from both the top and bottom.

  Bottom fluorescence   Avg Top Fluorescence   Avg
0.1 uM 2599 2245 2422 4392 4672 4532
1 uM 2385 2391 2388 4397 4345 4371
10 uM 1990 2082 2036 3824 5105 4464.5
100 uM 2023 2093 2058 3408 3574 3491

4. A blank was mistakenly not included so these values should not be considered as accurate or reliable. Fluorescence also seemed to decrease as the concentration of liposomes increased, which is odd but again, the results are not reliable. The experiment should be repeated with a new batch of liposomes.

September 29, 2016
Present: Ina, Angel

Task: Perform a permeability test on bEND3 cells seeded onto 0.4 µm PET transwell filters using Carboxyfluorescein

  1. 2 mL 0.1% BSA in sterile PBS solution was prepared inside the hood by adding 2 µL of BSA to 2000 µL of sterile PBS.

  2. A 1:50 solution of 0.625% Carboxyfluorescein (CF) in PBS was prepared by adding 4 µL CF in 200 µL sterile PBS. The CF was kindly given by John from the Naus Lab.

  3. The bEND3 cells seeded on the transwell filters last September 27 were used for this experiment. Media from both the top and bottom compartments were aspirated.

  4. The transwell inserts were moved aside with sterile tweezers to an adjacent empty well. 600 µL of 0.1%BSA + PBS solution was added to each well. Note that when working with transwells, one must allow some spacing on the plate for holding transwells while changing media. Alternatively, one can use a different plate to hold the inserts, in the event that we want to utilize all the 24 wells in one plate. Though, it’s probably better to just leave spaces in between wells (i.e., only seed cells and utilize alternating columns on the 24-well plate) since we’ll be doing a fluorescence assay and we don’t want the signal to overlap.

  5. 100 µL of the CF solution was added to each insert, and the insert was placed back inside their respective wells.

  6. The plate was left to incubate, and 100 µL samples from the bottom well were taken every 10 minutes for 30 minutes and transferred into a clear, flat bottom 96-well plate. Every time a 100 µL sample is taken, 100 µL of BSA+PBS is added back in to maintain constant volume at all times.
    Note: Missed the 20 min interval insertion into the well containing its respective PBS+0.1%BSA. This is why there is no 30 min sample. Continued with insertions at the 30 min interval.

  7. The SkanIt Varioskan Flash microplate reader was used to detect fluorescence at an Ex/Em of 495/517 for 110 ms scans. Samples were spaced out to reduce any crossover signal from the other wells. A blank of PBS +0.1% BSA was included, and the CF on the top layer of the inserts were also sampled (at the last time point). The layout is below:

Notes: Seeding density can still be increased. The area of the insert is around 0.321 cm2 and the paper by Wuest, D. et al (2012) used 2.5 × 105 cells/cm2 seeding density

For reference, here is the September 27 seeding density log:

September 27, 2016
Present: Angel, Ina

Task: Seed bEND3 cells onto transwell filter membranes

  1. 600 µL of media was loaded into the 2 wells of the 24-well plate (as per Corning media suggestions)

  2. BD Falcon 0.4 µm PET membranes were placed into the 2 wells and were left to perch on the 24-well plate

  3. 100 µL of media was added into the inserts

  4. The plate was allowed to incubate at 37 C for 1 hour in order to prime the system, as per suggestion by Corning

  5. bEND3 cells (P14) were counted using a hemacytometer

  6. A density of 4.67x104 cells (well A1) and 4.67x105 cells (well A3) were seeded (seeding density is based on an optimized seeding density suggested by Corning. The seeding density seemed reasonable when confirmed with papers by Guanglei Li., et al)

  7. Cells were left to incubate and grow overnight

The data obtained is below.
Note: no error bars because there were no replicates

Oct 3rd, 2016

Cells are seeded again
Conditions are 75000 cells, 150000 cells, 225000 cells

Oct 4th, 2016

The following protocol is used:

  1. 7 mL 0.1% BSA in sterile PBS solution was prepared inside the hood by adding 7 µL of BSA to 7000 µL of sterile PBS.

  2. A 1 µM Sodium Flourescin is made by first weighing 2.8 mg and adding 5 mL milipore water to that to make 1.382 mM sodium flourescin, then filter sterilizing using syringe filter and then adding 0.7 µL of this to 1000 µL PBS.

  3. The bEND3 cells seeded on the transwell filters last October 2 were used for this experiment.

  4. The transwell filters were moved to adjacent wells using sterile tweezers while having media inside them

  5. Media inside wells in 24 well plate was aspirated

  6. 600 µL of PBS+BSA solution is added to each well

  7. Each transwell is held by tweezers, and while its being held, media is aspirated from inside the transwells and then transwells are submerged into PBS+BSA into each well

  8. 100 µL of Sodium flourescin is added

  9. After each 15 minutes from time of adding, transwells are taken out, 100 µL sample of PBS+BSA is taken out, 100 µL of PBS+BSA is added to each well after all samples are taken out

  10. The SkanIt Varioskan Flash microplate reader was used to detect fluorescence at an Ex/Em of 460/515 for 110 ms scans. Samples were spaced out to reduce any crossover signal from the other wells. A blank of PBS +0.1% BSA was included, and the CF on the top layer of the inserts were also sampled (at the last time point). The layout is below:

Value 1 2 3 4 5 6 7 8 9 10 11 12
A 0.0814002   0.0821304   0.0882642   0.0903729   0.126197   0.101983  
B                       0.135254
C 0.0844324 0.0641084   0.0762362   0.0917187   0.11955   0.11178  
D                       0.144437
E 0.0977516   0.0818574   0.10386   0.0861179   0.12078   0.108329  
F                       0.14746
G 0.0713509   0.0757797   0.140754   0.158815   0.175585   0.207088 0.148353
H   0.172672   0.133077   0.203673   0.220212     12.164 12.2061

Note: At t=~9 min, two new transwells were added, and PBS+BSA was added to each of the new two wells, and sodium flourescin was added to their tops

As a result 150000 cells was used for future experiments.

October 8, 2016
Present: Ina, Angel, Amir

Task: Obtain baseline fluorescence measurements for the top fluor cholesterol liposomes

1. The molar concentration of the new batch of liposomes are calculated to be 1.85x104 µM. This was calculated assuming 100 nm diameter (without having sized the liposomes by DLS yet) and a concentration of 18.9 mg/mL. The calculations can be found at at this link

2. A serial dilution is prepared to obtain 100 µM, 10 µM, 1 µM and 0.1 µM concentrations. Dilutions were performed in the dark. 100 uL of sample in duplicates were added to the wells. The configuration can be seen in (3.)

Initial Concentration (µM) 1.85E+04      
Desired Concentration (µM) 0.1 1 10 100
Final Volume (µL) 350 350 350 700
V1 35.00 35.00 35.00 3.78
Volume needed to add 315.00 315.00 315.00 696.22

3. The fluorescence was read using the Varioskan SkanIt plate reader at the Hallam lab. An ex/em of 495/507 was used, but the results obtained did not make sense. The PBS blank had a fluorescence intensity higher than most of the samples. The fluorescence intensity seemed to increase with increasing dilutions.

Fluorescence assay on new batch of liposomes (Ex/Em 495/507):

Value   1 3   4 5 7 8
    trial 1 trial 2 average average minus blank blank1 blank2 average blank
A 100 uM 5017.63 4883.29 4950.46 -302.535 5265.44 5240.55 5252.995
C 10 uM 5065.09 5204.63 5134.86 -118.135      
E 1 uM 5182.62 5240.69 5211.655 -41.34      
G 0.1 uM 5273.54 5336.36 5304.95 51.955      

4. Repeated the experiment with new samples, using ex/em of 560/580 based on the suspicion that the ex/em should be at the red region. The rationale for this was that liposomes are visually emitting a red colour. Max from the Naus lab has also expressed that seeing a red colour should probably have an emission of red.

Value   1 2 3 4 5
A 100 uM 5.03649   5.76979 blank1 3.63866
C 10 uM 4.07231   4.27246 blank2 4.39127
E 1 uM 4.23701   4.33702 blank3 4.66974
G 0.1 uM 5.10263   5.39044 blank4 5.22693

5. Attempted fluorescence scanning and set the excitation wavelength at 530 nm, incrementing the emission at 5nm starting from 535 nm all the way to 615 nm. Scanning results seemed to be also very varied, and duplicate fluorescence units aren’t even very close to each other. After discussion with Amir, we agreed that the liposomes should somehow be sheared or dissociated since the Top Fluor cholesterol is embedded in the bilayer.

6. We decided to shear the liposomes by vortexing each tube (of 100, 10, 1, 0.1 uM liposomes in PBS) for ~30 seconds each. Fluorescence scanning was attempted again, but this time at a fixed excitation of 495 nm, varying the emission wavelengths from 507 nm to 531 nm at 2 nm intervals. The rationale for switching back to an excitation of 495 nm is the fact that during Cellomics high-content screening, we used an ex/em of 495/507 and the imaging worked perfectly fine.

It is likely that there is some sort of dye in the solution that colours the liposomes to be red, and that the fluorescence is safely embedded in the liposome bilayer so we can’t see this fluorescence.

Figure 1: Emission spectra at 495 nm excitation. Erroneous data and outliers were removed.

Figure 2: Fluorescence intensity count as a function of concentrations shows linear relationships for emissions 515 nm- 531 nm. The slope of the curves decrease as the emission is increased. A constant 495 nm excitation is used, and non-linear data were removed (below 515 nm emission).

Mini Experiment 4

Experiment 3