Cell Culture

Experiment 1:

Effect of EGF Ligands on Endocytosis Through U251 Cells


Note: U251 cells are not ready yet, but b.END3 cells are ready, as a result passaging b.END3 cells into 8 wells in 24 well plate such that seeding confluency would be 50%. For U251 cells, each replicate will come from a different flask and they will be seeded at 90%. Liposomes well be added on day 2 (day 0 is today).
Incubation time for liposomes will be 2 hours, then the cells will be fixed. Liposomes with EGF will be added to u251 cells at 4 different concentrations, liposomes without EGF will be added to all cells at same concentrations. Results will be measured using Cellomics.

202 live cells in 7 squares, 6 dead cells, 2ml total volume of cells, b.END3

30uM Amacon centrifugation filters: use to separate, spin down before DLS

U251 flask 1 count 203 live, 3 dead, 12 squares, tot vol 2 U251 flask 2, 224 live, 4 dead, 9 squares

Seeded 9 wells from flask 2, and 6 from flask 1 because of difference in cultures.

bEND3 bEND3   U251 f2 U251 f2 U251 f2
U251 f1 U251 f1 U251 f1 U251 f1 U251 f1 U251 f1
U251 f2 U251 f2 U251 f2 U251 f2 U251 f2 U251 f2

Seeding for U251 did not look good (center had more cells). New suggested sequence: Seed 1, Seed 2, then mix

  1. Clockwise for 5 seconds
  2. Counter-clockwise for 5 seconds
  3. Left and right for 5 seconds
  4. Up and down for 5 seconds

Then continue seeding.


This run will be done in accordance to the original cell culture experiments plan, meaning it will only have U251 cells. Astrocytes are not used because they are not available, however an increase in endocytosis between EGF having liposomes and the negative control liposomes would mean EGF has got a positive effect. In addition to examining effect of EGF on endocytosis, concentration of liposomes added and time of incubation of liposomes will also be titrated. 16 wells will be taken for concentration titration, for these wells, incubation time will 2 hours and 4 different concentrations of liposomes with and without EGF will be added. 8 wells will be used for examination of time of titration, where a single concentration of liposomes is added to all wells, and 4 different time points (different than 2 hours) will be examined. These will be 0.5 hours, 1 hour, 3 hours and overnight. Seeding for all wells will be 70,000 U251 cells per well. Passage 6 is used for this experiment.

Day 0 (Aug 29th): Flasks 1-1,1-2,2-2 are used for this experiment. Cells were washed, trypsinized for 2 minutes, pooled, centrifuged, resuspended and counted. 217 live cells, 3 dead cells in 8 squares was counted, from a total volume of 6mL. Seeded 0.129 from cells into 0.371 mL media, used Ina’s method for shaking each two seeded. Tomorrow liposomes are going to be added.

Day 1 (Aug 30th): Calculations for liposome concentrations were carried out in the excel file. Concentrations used are going to be 0.1-1-10-100 µM phospholipid and concentration used for time dependent experiment is going to be 1µM phospholipid.

Protocol is as follows:

Preparation of liposomes: need 2 syringes, 2 syringe filters, 8 15mL Falcon tubes, media, liposomes

  1. Label tubes Liposomes+EGF and liposomes
  2. Attach filter into end of syringe
  3. Take out plunger
  4. Add 1599 µL media and 11µL liposomes with EGF (mix liposomes by tapping the tube), for liposomes without EGF take
  5. 6µL and add 1744 µL media
  6. Bring syringe above the tube and push plunger back inside, ensure the syringe is located such that its contents would flow into the tube
  7. Add media to tubes for liposomes + EGF and without EGF as shown:

Liposomes + EGF

Concentration aims (µM phospholipid) 0.1 1 10 100
Number of wells for each condition 2 10 2 2
Volumes of each concentration needed (µL) 1100 5500 2000 1610
Dilution needed 110 550 200 11.0
Volume of media added 990 4950 1800 1599


Concentration aims (µM phospholipid) 0.1 1 10 100
Number of wells for each condition 2 2 2 2
Volumes of each concentration needed (µL) 1400 1400 1400 1750
Dilution needed 140 140 140 6.0
Volume of media added 1260 1260 1260 1744.0

Adding liposomes to cells:

  1. Aspirate media for each condition using P1000 at 600, slowly
  2. Add media+liposomes for that condition
  3. For Liposomes +EGF at 1µM, aspirate 4, add media+liposomes, then another 4, then last 2 (leave this concentration to last)
  4. Plate should be configured as follows:
0.1 Liposome+EGF-1 1 Liposome+EGF-2hr-1 10 Liposome+EGF-1 100 Liposome+EGF-1 1 Liposome+EGF-3hr-1 1-Liposome+EGF-0.5hr-1
0.1 Liposome+EGF-1 1 Liposome+EGF-2hr-2 10 Liposome+EGF-2 100 Liposome+EGF-1 1 Liposome+EGF-3hr-2 1 Liposome+EGF-0.5hr-2
0.1 Liposome-1 1 Liposome-1 10 Liposome-1 100 Liposome-1 1 Liposome+EGF-3hr-3 1 Liposome+EGF-1hr-1
0.1 Liposome-1 1 Liposome-2 10 Liposome-2 100 Liposome-2 1 Liposome+EGF-3hr-4 1 Liposome+EGF-1hr-2

Fixing Cells: Have a tube with 15mL PBS,
For conditions where only 2 wells are fixed:

  1. Take media out with P1000, add to waste beaker
  2. Add 200µL PBS to each well (add against wall)
  3. Take out PBS
  4. Add PFA with hoechst, time for 15 minutes
  5. Take out PFA, wash with PBS three times

The results of this experiment were produced through imaging with Cellomics. They were positive, in the sense that the 100 liposome EGF+ was endocytosed but the 100 liposome EGF- was not, in addition, the lower concentration liposomes did not show endocytosis, therefore, the original purpose of experiment B1 is achieved. However, in order to have results that we can strongly rely on for experiment B1, more replicates for this condition will be needed. Furthermore, the time titration experiments will be repeated with 100 µM Liposome EGF+.

Summary of results:

Ring is area around the nucleas, average intensity means intensity is averaged per area of each ring, mean points to the average for all cells in each well.


Day 0 (Sep 12th): Two U251 P10 cultures were pooled (resuspended in 2mL) and counted, then seeded into 24 well plate. Count: 226 cells in 3 squares, 2 ml total, Seeding 23 ul in 477 ul (70,000 cells per well) The following layout is used:

0.5 hr-1 2 hr-1 2hr EGF- -1   4hr -1 6hr -1  
0.5 hr-2 2hr -2 2hr EGF- -2   4hr -2 6hr -2  
  2hr -3 2hr EGF- -3       Just in case seeded
  2hr-4 2hr EGF- -4       Just in case seeded

Day 1:
Preparation of liposomes:

  1. Add 37.5 µL of liposomes+EGF to 5463 µL media
  2. Add 8.5 µL of liposome-EGF to 2491 µL media
  3. Prepare filter sterilizer (take out plunger, put syringe filter on, put over 15 mL tube) and put liposome with media through filter (by adding plunger), repeat for both cases

Adding liposomes to cells:

  1. Take out media with P100, after each two, add liposomes, start from 6 hr samples and move back
    2:24 time of start of incubation

Fixing cells: Have a tube with 15mL PBS, add 0.9 µL Hoechst to 6 mL pfa For conditions where only 2 wells are fixed:

  1. Take media out with P1000, add to waste beaker
  2. Add 200µL PBS to each well (add against wall)
  3. Take out PBS
  4. Add 400ulPFA with hoechst, time for 7.5 minutes @ 37°C
  5. Take out PFA, wash with PBS three times


Mini Experiment 3

Mini Experiment 4