Hela cells

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					                                    Maintenance of Hela cells
      A schedule of cell maintenance, feeding and passaging, should be developed to maintain
       appropriate cell density, nutrient concentration and pH levels in cultures.
      Cells are best passaged when they are growing logarithmically, at 70 to 80 % confluency
      This is an example of the schedule used for routine maintenance of Hela cells. Cells are
       maintained on Mondays and Fridays, but any combination of 3 nights/4 nights schedule is okay.
       Volumes given are for a T25 flask.
      Avoid trypsinizing the cells you are carrying forward too often, since this selects for trypsin-
       resistant cells and causes the passage count to increase too quickly. Cells should be carried for a
       maximum of 25 passages (from original stocks). Always do your experiments in a 20 passage
       window.
Monday:
-Inspect the culture for contamination. Cells should be 70-80% confluent.
-Prewarm Hela media and TE 0.05% in the 35C water bath for 15-20 min.
- Aspirate old medium from the flask with a sterile Pasteur pipet.
- Wash the cells with 5 ml of calcium-magnesium free-phosphate buffered saline (CMF-PBS) to
remove any residual medium. (~ 15 sec). Aspirate with a sterile Pasteur pipet.
- Add 1 ml 0.05% trypsin-EDTA (TE) to the flask and evenly disperse over surface by rocking. After
thawing, the TE can be stored at 4 C for up to 2 weeks. Do not freeze-thaw.
-Place the flask into the incubator with the cap screwed on tightly. Take the cells out every 2 minutes
to check the progress of detachment. Make a note of the time it takes.
- When the cells are detached, add 5 ml new media and rinse the surface of the flask. Pipet up and
down to dissociate cell clumps a few times. Check briefly under the microscope to confirm that cells
are dispersed in a single-cell suspension (>80%) and that any cell clumps have about 5 cells or less. If
not, pipet up and down 3 more times max. Minimize foaming since it is very hard on the cells.
-Transfer a small amount (<0.5ml) to a microfuge tube as a sample to get a cell count.
- Remove 50 l cell suspension to mix with 450 l 0.1% Trypan Blue (TB) for counting.
-Calculate the volume of cell suspension required to get the desired seeding density, depending on the
number of nights you intend to leave them before the next passage. If volume is <0.2 ml, make a
dilution of the cell suspension in a sterile tube.
-Add the appropriate volume of cell suspension to the new flask (2104 cells/cm2  25 cm2 )and add
enough fresh media to make 5 ml total.
-First mix the cells with the flask upright. Then lay it down and swirl in a figure 8 pattern. It's
important to get the cells evenly distributed.
-Return the flask to the incubator.
Wednesday:
Cells are fed with DMEM-10%FBS.

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- The culture is inspected under the microscope to ensure no contamination is present and
       cells look "normal".
-Aspirate old medium from the flask with a sterile Pasteur pipet.
- Add 5 ml is new medium.


Friday:
-Treat cells similar to Monday, but seed at 4104 cells/cm2. If the cells are more than 100% confluent,
adjust the density they are seeded at.

COUNTING CELLS
Mix 450l 0.1% Trypan Blue in PBS(dye) with 50 l cell suspension. Mix gently but thoroughly.
Transfer 10 l of the above mixture to counting chamber (with cover glass on top)
Count number of cells within "counting squares"
Example calculation:
      counts = 83, 85
      average = 84
      84/4 x 104 x 10 (dilution factor) = 1.05 x 106 cells/ml
      Total # cells = 1.05 x 106 cells/ml x 5ml = 5.25 x 107 cells

THAWING CELLS
It's important to have everything ready before retrieving the vial, to minimize the time the thawed cells
are exposed to high concentrations of DMSO.
 Dispense 10 ml of media into a sterile 15ml tube and warm in a 37oC waterbath.
Remove the freezing vial from liquid nitrogen and place on dry ice until everything is ready.
 Thaw the vial in a 37oC waterbath by gently agitating. The O-ring and cap should be kept out of the
water to prevent contamination. Remove the vial from the water when as soon as the contents are
thawed.
 After thawing, spray the entire vial with 70% EtOH.
Remove the cell suspension from the vial with a 1ml pipet and added dropwise to the 10 ml pre-
        warmed medium.
Pellet the cells by centrifugation at 1000 rpm for 5 minutes.
Aspirate the old medium with a Pasteur pipet. Do not disturb the cell pellet but attempt to remove
      all the old medium.
 Disperse the cell pellet by gentle tapping and flicking of the tube to prevent cell clumping when
      medium is added.
Add fresh medium (5ml) slowly to the tube. Resuspend the cells by gentle pipeting.
Add the cell suspension to a T25 flask and place in the incubator overnight.


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The next day, aspirate the old medium and replace with fresh medium. Passage cells when they
reach about 80-90% confluency.

FREEZING CELLS
To get approximately 5-6 vials for freezing, grow a T-150 flask to 70-80% confluency.
Warm a 5 ml aliquot of TE (0.05%) and Hela medium.
Prepare freezing medium (7.5% DMSO in normal media) by adding 1.5 ml DMSO to 18.5 ml HELA
medium in a 50 ml tube. Filter through a 0.22m syringe filter and place on ice.
Prepare 10-15 vials by setting them up and labeling. Indicate the cell type (Hela), the passage number,
the date, lab book information, and number of cells (1106).
Aspirate the old medium from the flask using a sterile Pasteur pipet.
 Wash the flask with 15 ml of CMF-PBS to remove any residual medium. (~ 15 sec). Aspirate using
a sterile Pasteur pipet.
Add 5 ml TE to the flask and rock back and forth so the surface is completely covered with TE.
Place the cells in the incubator and view periodically until they are detached.
Add 15 ml new media and rinse the surface of the flask. Pipet up and down to dissociate cell clumps
6 times. Check briefly under the microscope to confirm that cells are dispersed in a single-cell
suspension (>80%) and that any cell clumps have about 5 cells or less.
-Transfer a small amount (<0.5ml) to a microfuge tube as a sample to get a cell count.
- Remove 50 l cell suspension to mix with 450 l 0.1% Trypan Blue (TB) for counting.
Pellet the cells by centrifuging at 800 rpm (100 g) for 5 min.
While the cells are centrifuging, determine the cells/ml. Calculate the total number of cells in the
tube and then the volume of freezing medium to add to give 5106 cells per ml.
Aspirate the old medium with a Pasteur pipet.
Disperse the cell pellet by gentle tapping and flicking of the tube and then resuspend in the
calculated volume of pre-chilled freezing medium.
Dispense 1 ml of the cell suspension into each vial and place on ice.
Put the vials in a styrofoam box with a lid and store in -80oC freezer overnight. Transfer the vials
to liquid N2 the next day.

MEDIA PREPARATION
Dulbecco's modified Eagle's medium with 10% fetal bovine serum (DMEM-10%
FBS):
DMEM: Available from Biochem Stores (Invitrogen cat. # 11965-092, 500 ml bottle).
Fetal bovine serum (FBS): Available from Biochem stores (Invitrogen cat. # 10437-028). Stored at
-20C. After thawing it needs to be aliquoted. To thaw: Remove the serum from the freezer and place
it overnight in the fridge. Transfer to 37C water bath. Agitate from time to time in order to mix the

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solutes that tend to concentrate at the bottom of the bottle. Do not keep the serum at 37C any longer
than necessary to completely thaw it. Set up 50 ml sterile tubes in the hood. Label FBS with the expiry
date and loosen caps. With a sterile 25 ml pipet, carefully aliquot 27 ml each and seal the tubes with
parafilm. Store the aliquots at -20C. Make an entry in the "cell culture reagents" section of the Cell
culture book #1 to avoid having two lots going at once (serum is very expensive!)
To make Hela media:
Thaw a 27 ml aliquot of fetal bovine serum in a beaker of warm water, or overnight in the fridge.
When thawed, invert to mix and keep on ice or at 4C until needed.
 Add 25 ml serum to a T-75 flask and add 225 ml DMEM, mix well.
Label your media with the date and a "media number". We keep track of all media made in the
"media log" section of Cell Culture Book 1.

Calcium-magnesium free- phosphate buffered saline (CMF-PBS):
There should be a supply on hand, but here is the recipe:
Per litre:
0.2 g KCl
0.2 g KH2PO4
8.0 g NaCl
1.15 g Na2HPO4
Store in 500 ml bottles. To recycle bottles, wash thoroughly with water and then MQ water. Do not
put soap in PBS bottles.
- Sterilize by autoclaving.

TE (TRYPSIN-EDTA):
TE (0.05%) is obtained from Biochem stores (Invitrogen) cat #25300-054, 100 ml bottle. Store at -
20C. After thawing it must be aliquoted. Thaw a bottle in the fridge overnight, or in a 37C water
bath. Mix occasionally and remove as soon as it is thawed. To aliquot, set up twenty 15 ml tubes in
the hood. Label TE .05% and indicate the expiry date. Loosen caps and carefully add 5 to each tube
and tighten. Make an entry in the "cell culture reagents" section of the Cell culture book #1.

 Trypan-Blue dye (TB):
mix 0.4% TB with PBS to have a final concentration of TB in 0.1% for counting
      cells.




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