Procedures I:
Volumes are given for a 75 cm2 flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes. Corning® T-75 flasks (catalog #430641) are recommended for subculturing this product. Sterile technique is necessary for all procedures in this section.
Remove and discard culture medium.
Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
Add appropriate aliquots of the cell suspension to new culture vessels.
Incubate cultures at 37°C.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:8 is recommended
Medium Renewal: Every 2 to 3 days (ATCC)
Procedures II:
Make sure oscilloscope is getting readings properly from DDS signal generator.
Fill a 2 steel crucibles with 30mL of distilled water.
Next, find a cryogenic vial and fill it with distilled water.
Place one crucible in a -86ºC freezer, place the cryogenic vial into the cryogenic freezer, and put the other into a regular freezer.
Wait 30 to 45 minutes to allow all of the ice to completely freeze.
Connect the probes to the crucibles for 10 minutes each at 20-25MHz.
Assess which method of freezing had the most damage and report the results.
Procedures III:
Note that sterile technique is used until step 9.
Discard old medium for 4 flasks of cells (3 trial groups and 1 control).
Wash cells with 3mL of Phosphate Buffered Saline (PBS), then discard it into a waste beaker.
Add 3mL of 0.25% Trypsin-EDTA solution to the flasks and incubate at 37ºC to facilitate dispersal.
Once cells have dispersed, add 10mL of complete growth medium to neutralize the Trypsin.
Move the solution into a 10mL sterile centrifuge tube and centrifuge for 5 minutes at 250rpm. Once a pellet has formed, discard old medium and add 10mL of fresh medium to resuspend the cells.
Once cells are resuspended, move the solution into a 50mL sterile centrifuge tube and take a 2mL sample for cell count.
Add 1mL of Trypan-Blue solution into the tube and mix delicately. Take 10µL of the complete cell solution and add it to both sides of the hemacytometer.
Count cells. Record cell count in log notebook.
Divide cells into 4 tubes by volume but make sure cells are dispersed before transfer.
Prepare oscilloscope and DDS frequency generator.
Label centrifuge tubes “Control,” “Trial 1,” “Trial 2,” and “Trial 3.”
Take the tube labeled control, pull a 1mL sample from it, and put it into the crucible.
It was observed during Procedures II that the -86ºC freezer worked the best for the purposes of this experiment. Only two crucibles were able to tested at a time due to the nature of the DDS frequency generator.
Take the tube labeled trial 1, pull a 1mL sample from it, and put it into the crucible.
Put both crucibles in the -86ºC freezer for 15 minutes.
Once the 15 minutes has elapsed, connect the probes to trial 1 and thaw the control group.
During the 5 minutes that trial 1 is occuring, take the cell count of the control, but be very wary of the time.
Once the control is counted, wash the hemacytometer with a 1:10 bleach to dH2O to bleach mixture, then rinse with dH2O.
Take the cell count of trial 1 and compare it to the control. Once trial 1 is counted, wash the hemacytometer with a 1:10 bleach to dH2O mixture, then rinse with dH2O.
Wash crucibles thoroughly with a 1:10 bleach to dH2O mixture, then rinse with dH2O.
Repeat steps 12-19 for trials 2 and 3. Try to get pictures of cell count if possible, however due to the nature of the experiment, it is very hard to get pictures.
Clean up work station. Discard all biological waste into biohazard, neutralize all liquid waste with bleach before pouring it down the sink, and keep gloves goggles, and aprons on until the cleanup is completed.