Tissue Culture Procedures

Tissue Culture Procedures:

REMEMBER:

·  Use your own media and buffers (labeled with your initials and date). DO NOT SHARE MEDIA OR BUFFERS

·  Use your own autoclaved Pasteur pipets

·  Aliquoted material (e.g. Calf serum, Pen-Strep, Glu, Trypsin) is general use

·  Always tape up tightly bags with petri dishes to maintain sterility

CHECKLIST (when you are finished with cell culture)

·  water bath: OFF

·  centrifuge: OFF

·  pipet aid power: OFF

·  hood: OFF and UV light on

·  vacuum line: OFF

·  incubator: reading 5% CO2 and 37C

PROPER CARE OF TISSUE CULTURE HOOD

·  Standby mode:

·  main switch and power switch (for plugs inside hood): OFF

·  UV light: ON

·  Blower: OFF

·  When using hood...

a)  Switch UV light to fluorescent light

b)  Turn blower on

c)  Hood should be turned on at least 15 minutes prior to use

d)  Wipe surface with 70% ethanol (see “Preparation of 70% Ethanol”)

PREPARING 70% ETHANOL SOLUTION

The solution for wiping down the cell culture hood and any items that go into the hood (e.g. media, thawed aliquots, etc.) is 70% by volume ethanol and 30% Milli-Q water.

You can use clean glassware to make the solution. (e.g. in a 200 ml flask, add 140 ml ethanol and 60 ml water).

PROPER CARE OF NIKON MICROSCOPE (Cell culture microscope)

IMPORTANT: Once microscope is turned on, LEAVE ON UNTIL END OF DAY!!

(Turning on and off multiple times reduces lifetime of bulb)

·  Standby mode: Leave switch on low (just BEFORE shut off, i.e. below #2 on intensity level) when you are finished looking at your sample. Turn completely off only at the END OF THE DAY.

·  AVOID setting the light to the RED ZONE. This also reduces the lifetime of the bulb.

·  At end of day, turn off and put dustcover on microscope.

USING A HEMACYTOMETER TO COUNT CELLS

Read instructions. Ask if in doubt.

STERILIZING PIPETS, GLASSWARE FOR TISSUE CULTURE

Read instructions. Ask if in doubt.

PROPER CARE OF CRYOGENIC CELL STORAGE

a)  Always wear safety glasses and gloves when opening the storage tank

Q: How often do I have to add liquid nitrogen?

A: We will probably have to fill it once every few months. It is always a good idea to check. If you notice the level is low when you take a vial out to thaw, it is time to fill.

Q: Where do I get liquid nitrogen?

A: There are several sources that we get our liquid nitrogen from. The first place to go is Jonathan Lee’s lab in Chemistry. His lab is in Rm 394 in Chemistry (ph: 3-2477). Ask for Tyler or Marcel. You may also want to bring over a bottle of beer – I think we are up to two bottles now!! ;-) The other place is to go up to the 6th floor and ask the Hansen/Cooper lab. It is actually located on the 7th floor.

Q: Do I bring the whole cell tank up?

A: NO! Bring a dewar to fill the tank, even if it is up to the 7th floor. Remember that the storage tank is very fragile and you don’t want to be rolling it around everywhere.

PROPER CARE OF INCUBATOR

The centrifuge should always read 37C and 5% CO2. It is a good idea every few months to check the actual CO2 level with a Fryrite reader. They have one on the 7th floor.

Q: Whom should I contact if there is an emergency with the incubator?

A: Our sales rep is Mary Murphy at Biological Equipment Specialties (ph: 1-800-335-3155). It is best to leave her a message detailing the nature of the problem.

Q: What happens if the CO2 tank level is low?

A: Periodically you should check the level of CO2. Our incubator has an automatic switch on the back that will switch to the second tank when the first tank is empty. This is a good time to call AGA gas and order a new CO2 tank. They will come the next day. Call Bob Zinnani (978-439-1364 vmail) and leave a message. We have a blanket Purchase order with them, so there should be no problem.

PROPER USE OF THE CENTRIFUGE

Our centrifuge currently needs repair, but it appears to work okay.

Check with Mark on how to use the magic touch.

Q: Whom should I contact if there is a problem with the incubator?

A: Again, this is Mary Murphy (ph: 1-800-335-3155).

PREPARATION OF CELL CULTURE MEDIUM

a)  Warm all solutions in the 37C water bath prior to use
3T3 Fibroblasts, materials needed to thaw:

i)  500 ml bottle of DMEM high glucose

ii)  Aliquot (50 ml) of bovine calf serum

iii)  Aliquot of Pen-Strep/Glutamine (5 ml each)

b)  Rinse all bottles and tubes with 70% ethanol and wipe before bringing into hood

c)  Add 50 ml calf serum and 10 ml Pen-Strep/Glutamine to 500 ml medium. This yields a 10% CS solution. The bottle should be labeled as complete containing 10% CS with your name and date. The amount of calf serum should be written on the bottle in two separate places to ensure that no mistake in identity will be made in the future.
Mix in media bottle (gently swirl bottle or use pipet aid to mix).
Note the color of the media after calf serum is added. Mark the bottle appropriately: 10% CS-DMEM and date.

Q: How long can I use the media??

A: If you are using older media (e.g. about 3 weeks old), use fresh glutamine – this must be added once every few weeks. Check clarity of media before each use. Complete media should not be stored for more than a month or so.

PROPER CARE OF 3T3 FIBROBLASTS:

Thawing Trypsin:

Thaw trypsin in water bath just to the point where it thaws. Any additional time in the 37C water bath will inactivate the action of the trypsin.

Seeding Cells:

If from frozen cells...

Pipet 15 ml complete media (10% CS-DMEM) into 250 ml flask (T75). If using 100 mm culture dishes, 10 ml of medium is sufficient. If using frozen cells, the media must first be warmed in the incubator for at least 15 minutes; quick-thaw cells in water bath and transfer to flask/dish with warm media. Change the media again after 24 hours – this gets rid of excess DMSO.

If seeding from other flask or dish...

LABEL plates: (Your initials, Cell type, Passage #, Date)

a)  Suction off medium in seed flask

b)  Add 10 ml of phosphate buffered saline (PBS) (15 ml, if flask) and aspirate using sterile Pasteur pipet and suction. The PBS rinse is necessary because the trypsin will be inactivated by the remaining Calf Serum.

c)  Add 2 ml of 0.05% trypsin, 0.53 mM EDTA, just to wet the surface; place plate at 37C for 2-5 min (until cells have rounded up and have begun to detach). Shake plate gently to release the cells from the plastic surface

d)  Look in microscope. If cells are rounded, they are ready to fall off. Shake flask/dish and tap to loosen the cells. Keep checking under microscope

e)  Suspend cells in 10 ml media (serum inactivates the trypsin).

f)  Transfer to a 15ml or 50 ml sterile centrifuge tube

g)  Centrifuge the cells for about 10 minutes

h)  Aspirate gently the media, leaving some liquid above the pellet

i)  Resuspend the cells in the appropriate amount of media (depends on how dense you want to plate the cells)

j)  Add cells to plates that already contain 10 ml of media

k)  Before returning flasks to incubator, mix contents gently, and make sure that no media is spilling outside of the dish. If there is, clean by aspirating with a Pasteur pipet

Feeding Cells:

Cells should be fed at least once a week by aspirating off media carefully and adding fresh pre-warmed media.

Q: How long can I keep passaging these cells?

A: When the cells are thawed from a frozen vial, you can use them for about two months. After this, you will need to thaw down a new vial. Remember to keep track of the passage number! Remember also that each cell line is different and there may be a different procedure for their maintenance.

Freezing down 3T3 Cells:

a)  Aspirate media from a dish.

b)  Rinse with PBS

c)  Aspirate off with a sterile Pasteur pipet

d)  Add 2 ml trypsin to just wet plate surface

e)  Resuspend cells (with prewarmed complete media) in a total volume of 6-8 ml

f)  Count cells in a hemacytometer. (The number in one 4x4 square (there are eight of these on a hemacytometer) divided by 10-4 gives the number of cells per ml. Multiply this number by the volume of the media. This number equals the total number of cells in the dish.

g)  Put cell solution in centrifuge tubes and spin down gently to pellet cells.

h)  Aspirate media supernatant

i)  Resuspend cells at 1.5 e6 /ml in DMSO media ( i.e. complete media + 10% media colume DMSO – dimethyl sulfoxide, also known as methyl sulfoxide). Use sterile DMSO.

j)  Transfer to vials (labeled with cell type, passage number and date) in 1 ml aliquots.

k)  Freeze at –70C overnight. Note: you must freeze immediately because DMSO is toxic to cells at room temperature.

l)  Transfer vials to cryogenic storage (AVOID the top two slots in the racks in case the level of liquid nitrogen drops)

GETTING RID OF WASTE

- used pipettes

- sharps