Lab 3
BCH/BIO 212
Cell Biology
Fall 2004
Culture of HeLa Cells
Reading:
The Immortality of a Cancer Victim Dead Since 1951 (article from 1976 Smithsonian magazine)
The cells we will be growing this week will be used for protein purification and analysis over the subsequent
three lab periods. The schedule is:
Lab 3: passage HeLa cells
Lab 4: lyse cells and use antibodies to purify one protein from the cell lysates (immunoprecipitation)
Lab 5: separate the isolated protein (from the antibodies) by size using gel electrophoresis (SDS-PAGE)
and transfer the separated proteins from the gel onto a membrane
Lab 6: use antibodies to detect the amount and determine the MW of the purified protein (Western blot)
The cells we will use are a widely used human cervical cancer cell line known as HeLa cells. These cells are
adherent (i.e. they stick tightly to the cell culture flask). Adherent cells are typically dislodged from their culture
flasks by treatment with trypsin (a protease that digests extracellular proteins involved in attaching the cells to their
substrate) and EDTA (a chemical that chelates divalent cations - e.g. Mg2+, Ca2+ - which are required for the function
of some cell adhesion molecules) before they can be passaged.
HeLa cells growth medium
DMEM (Dulbecco’s Modified Eagle’s Medium) --- list of components in DMEM was distributed in Lab 1
10% FBS (fetal bovine serum)
Glutamine
Penicillin/streptomycin (pen/strep)
Passage of HeLa cells
1. Wipe down the hood with ethanol before you start to work.
2. Warm DMEM+10% FBS medium, 1X trypsin, and PBS (phosphate-buffered saline) in the 37oC
water bath.
3. When solutions are warm, spray the bottles with 70% EtOH, wipe, and place in the hood.
4. Put a 15-mL conical tube and 4-5 sterile 10-mL pipettes in the hood.
5. Loosen the caps of all containers.
6. Get cells from the incubator. Observe them under the phase-contrast microscope and note their
appearance. Turn off the light on the microscope. Bring the cells to the hood.
7. Remove a sterile Pasteur pipette from the metal canister.
Be careful not to touch the other pipettes or the interior of the canister.
Pull the pipette straight out of the canister without touching it to any surface.
8. Place the non-pointed end of the pipet in the plastic vacuum aspirator tube.
9.
With the vacuum pump turned on, carefully aspirate the medium from the cells.
It is helpful to tilt the flask to remove the last portion of medium.
10. With a fresh pipette, place approx. 10 mL of sterile PBS in the flask on the side opposite the
cells.
1
11. Tilt the flask back and forth to wash the cells with PBS.
12. Use the same pipette (assuming it's uncontaminated) to remove the PBS from the cells.
13. Squirt the PBS into the liquid waste container.
14. With a fresh pipette, place 2 mL of 1X trypsin/EDTA in the flask.
15. Close the flask's cap, then tilt it to spread trypsin over the cells. Lay the flask down.
(You can incubate it in the CO2 incubator to speed the action of the trypsin.)
When the cell layer begins to slough off you will see holes in the monolayer with the naked eye.
The medium will take on a cloudy appearance.
When viewed under the microscope, the cells will be seen to round up and float.
This is your sign to go on to step 16 to stop the trypsin.
Do not keep trypsin on the cells for any longer than necessary or the cells will begin to lyse and die.
Be GENTLE with the cells through the rest of the protocol.
16. Tap the flask lightly against the surface of the hood.
17. Turn the flask upright, open the cap, then use a fresh pipette to add 8 mL 10% FBS/ DMEM.
Gently spray the medium down over the surface of the cells to dislodge them.
18. Draw the medium back up into the pipette, then gently spray the medium over the cell surface
again.
19. Repeat several times to dislodge most of the cells.
Don't do this more than 5 times or you will start to lyse the cells.
20. Use the same (uncontaminated) pipette to place the cell suspension into the 15 mL conical tube.
Close the cap of the tube.
21. Place the tube of cells into the CL-2 centrifuge.
Be sure to use the rust-colored adapters and a balance tube.
21. Spin for 6 minutes at 1000 rpm (setting of 1.0).
23. During the spin, get 4 Costar T-75 flasks. Label the top surface with the cell line (HeLa), the
passage number, the date, your group's initials, and the passage split.
24. When the spin is complete is complete, wipe down the tube with EtOH and return it to the
hood.
25. Use a sterile Pasteur pipet and the vacuum aspirator to remove most of the supernatant.
Leave several millimeters of medium over the top of the cell pellet - be careful not to dislodge the
pellet!
26. Use a fresh pipette to place 5 mL DMEM/10% FBS in the tube containing your cell pellet.
2
27. Use the same (if non-contaminated!) pipette to place 12 mL of DMEM/10% FBS in each
T-75 flask.
You can use the same pipette for all flasks as long as you don't touch it to anything non-sterile.
28. Gently aspirate the cells up and down (no more than 5 times) to resuspend your cells.
29. Use the same (non-contaminated!) pipette to place _____ mL of this cell suspension in each of
your T-75 flasks.
The final "split" will be the volume of cells you add to each new flask : the total volume of cell resuspension, i.e. ____________ (vol added/flask) : _________ (total vol cells) = ___________ split.
Example: Cells resuspended in 10 mls medium. 2.5 mls resuspension added to each flask.
Split = 2.5:10 or 1:4.
The total volume of medium (fresh medium + vol of resuspended cells) should be 12 - 15 ml/flask.
30 Close the lids of the flasks and tilt them back and forth to evenly distribute the cells.
31. Place the flasks in the incubator, then open the caps a half-turn and close the incubator.
When you close the incubator, sure to latch the inner glass door and close the outer door firmly.
32. Return media, unused pipets, etc. to their proper locations.
33. Wipe the hood down with EtOH and close the glass window (sash).
34. If you are the last person using the room, turn off the electrical outlets, blower, and light in the
hood.
35. Check that the:
microscope light is turned off
refrigerator door is closed
the water bath turned off
centrifuge is turned off
vacuum pump by hood is turned off
The woman from which the HeLa cells are derived was named Henrietta
Lacks, and she was a wife and mother of five when she died at Johns
Hopkins Hospital in 1951 at the age of thirty-one.
A sample of one of her cervical tumors was sent to George and
Maragret Gey who had been seeking a line of human cells that would
survive indefinitely outside the body for research purposes.
The tumor cells they received multiplied like nothing they had ever seen
before and soon the cells, dubbed “HeLa” in a truncated form of Lacks'
name, were being shipped to scientists around the world. The cells later
became a laboratory standard and have even been grown in space.
From: http://www.microscopyu.com/galleries/dicphasecontrast/helapclarge.html
3
Techniques of Cell Culture
1.
Wear gloves when working with cells. Although the gloves we use are not sterile, they protect your
cultures from bacteria-laden skin flakes which fall from your hands. They also protect you against any
possible pathogens in the culture.
2.
Do all cell work in a laminar flow hood. The laminar flow hood contains HEPA (high efficiency
particulate) filters which remove microscopic particles so that sterile air is blown down from above.
3. Before working in the hood, turn the blower on for at least five minutes to filter the air already in
the hood and establish laminar flow. Spray the work surface with 70% ethanol and wipe to remove
loose dust particles.
4. Before using bottles in the hood, wipe off residual water and spray them with 70% ethanol. Wipe
off to remove dust. Place bottles in the hood and loosen (don't remove) the cap.
5.
When using the hood, do all work over the smooth metal surface, not the grate. Do not pass
sterile materials outside of the glass - they will become contaminated!
6.
Do not pass your hands/arms over open containers in the hood. The air flow can blow loose
particles from your gloves or shirt into the container.
7.
There are two ways to deal with opening bottles in the hood. My preferred way is to open the
bottle and hold the cap in your hand, right (closed) side up, without placing it on the work surface.
Often you may not have enough hands to do this. It is also acceptable to place the cap, right side
up, on a clean area of the work surface. Do not hold or place the cap upside down - liquid on the lip
may drain into the cap and contaminate it.
8.
To open pipets, peel apart the plastic from the paper, like removing the peel from a banana. Insert
the pipet into the pipet aid. While holding the wrapper in one hand and the pipet aid in the other,
check to see that you can easily read the graduations on the pipette. Then pull the pipet straight out
of its wrapper without touching it to the non-sterile outside of the wrapper. Set the wrapper
aside. When you are finished with the pipet, place it back into its wrapper and set it aside. Discard
used pipets in the biohazard waste can.
Laminar flow hood
http://www.classiccare.on.ca/cleanroom.htm
4