Cell Culture


Mini Experiment 1:

Seeding Density Titration for 24 Well Plate

Purpose: Finding optimal seeding density for both b. END3 and U251 cells in 24 well plates.

Note: 24 well plate is going to be used only because 96 well transwell filters would be too expensive and we would need to waste most of it for our experiments. In addition, it is easier to use 24 well plates.

For this experiment, previous counts in maintenance logs are used and data from Corning’s website regarding well sizes are used.

Cells for this experiment are U251 seed high from Aug 5th, it is at 65% confluency. It is not very healthy as it hasn’t grown since yesterday, however, it should be fine for our experiment. B.End3 is also used from Aug 12th (flask 1), these cells are at 85% confluency and not healthy neither as they have not grown since yesterday neither.
Each condition has two replicates.
Calculations are done in an Excel file.

Protocol:

  1. Aspirate media from b.END3 flask
  2. Add PBS
  3. Aspirate media from U251 flask
  4. Add PBS
  5. Aspirate PBS from b.END3
  6. Add 1mL trypsin
  7. Aspirate PBS from U251
  8. Add 1mL trypsin
  9. Put both in dry incubator for 2 minutes
  10. Prepare hemocytometer while waiting
  11. Tap flasks to assure cells are dislodged and observe under microscope
  12. Add 2 mL media to U251 flask, transfer to 15mL falcon tube
  13. Add 2 mL media to b.END3 flask, transfer to 15 mL falcon tube
  14. Centrifuge for 5 min at 1g
  15. Aspirate media/trypsin from U251 tube, resuspend in 1mL media using 1mL pipette, take 0.1 mL add to small microcentrifuge tube
  16. Aspirate media/trypsin from b.END3 tube, resuspend in 1mL media using 1mL pipette, take 0.1 mL add to small microcentrifuge tube
  17. Take 50µL of tube for U251, add to new tube, then add 50µL of trypan blue, then mix 5 times, then take 50µL and add to hemocytometer, count
  18. Clean hemocytometer with ethanol and kimwipes
  19. Add count result to excel file
  20. Repeat step 17 and 19 for b.END 3
  21. Using new values, add media to wells in 24 well plate
  22. Add cells
  23. Put in incubator, do L shaped mixing for 15 times
  24. Clean hemocytometer
  25. Clean hood

U251 202 live cells, 4 dead, 18 squares B.END3 207 live cells, 7 dead, 8 squares

Format is as follows:

U251 first seeding U251 second seeding U251 third seeding      
B.END 3 first seeding replicate 1 B.END 3 first seeding replicate 2 B.END3 second seeding replicate 1      
B.END3 second seeding replicate 2 B.END3 third seeding replicate 1 B.END3 third seeding replicate 2      
           

Calculations

U251 90% 880000 22.5 39111.11111 37155.55556 96 0.32 0.2 9511.822222 47559.11111
  75% 660000 18.75 35200   24 1.9 0.5 70595.55556 141191.1111
b.END3 90% 1040000 22.5 46222.22222 43811.11111 96 0.32 0.2 11215.64444 56078.22222
  80% 828000 20 41400   24 1.9 0.5 83241.11111 166482.2222
                     
                     
  Confluency Area Number of live cells Volume Density Total Cells needed Volume needed Volume of media needed to be added    
U251 65% 16.25 226666.6667 1 226666.6667 70595.55556 0.311 0.19    
            74917 0.145 0.36    
            66592.88889 0.129 0.37    
                1.72    
                     
                     

Maintenance Log for b.End 3

Mini Experiment 2