CELL BIO LAB WEEK 10

Which cells are cultured this week?

NIH 3T3 cells, a mammalian cell line derived from mouse fibroblasts.

What medium are the 3T3 cells grown in?

DMEM+, Dulbecco's Modified Eagle's Medium.

What does the '+' in DMEM+ mean?

The medium contains 10% (v/v) bovine calf serum and 1% (v/v) antibiotic/antimycotic.

Why is bovine calf serum added?

It is a growth supplement: it carries growth-promoting factors and supplies defined and undefined components that meet the cells' metabolic needs.

Why is antibiotic/antimycotic added?

To prevent bacteria and fungi from growing in the culture.

What is in the 100X antibiotic/antimycotic solution?

10,000 units/ml penicillin, 10,000 micrograms/ml streptomycin and 25 micrograms/ml amphotericin B.

What are the incubation conditions for 3T3 cells?

37 degrees C and 5% CO2.

Why must CO2 be supplied to the incubator?

The medium's pH depends on a balance of dissolved CO2 and bicarbonate, so a CO2-bicarbonate buffered medium needs exogenous CO2.

Why are cell cultures so easy to contaminate?

The medium is full of nutrients and growth factors, even though it contains antibiotics and antifungals.

Why can one contaminated flask endanger the others?

The incubator's air circulator spreads the contaminants into other cultures.

What does it mean that 3T3 cells are adherent?

They attach to the bottom of the flask and grow as a single monolayer.

When must the cells be passaged?

At about 80% confluence, though they can be passaged earlier if needed.

What does 80% confluence mean?

80% of the surface of the culture vessel is covered with cells.

What does passaging involve?

Detaching the cells from the flask, diluting them with medium in a new flask, and letting them reattach to the new surface.

How do 3T3 cells attach to the flask?

They secrete collagen, fibronectin and other extracellular matrix proteins.

How do 3T3 cells attach to each other?

Through cadherins, calcium-dependent adhering proteins.

What does trypsin do during passaging?

It is a protease that cleaves the extracellular matrix proteins, releasing the cells from the dish.

What does EDTA do during passaging?

It chelates calcium, so cadherin contacts between cells come apart and the cadherins become sensitive to trypsin.

Why rinse with PBS before adding trypsin?

To remove the serum, which contains trypsin inhibitor, and the calcium, since PBS lacks calcium.

Why add BCS once the cells have detached?

With the added PBS it dilutes the trypsin, and the serum's trypsin inhibitor stops it.

What airflow do our cell culture hoods use?

Vertical laminar flow of air passed through HEPA filters.

What gets sprayed with 70% ethanol?

Everything that goes into the hood, including your gloved hands.

Where may materials sit inside the hood?

Only on the solid surface of the hood.

What must be true before you start a protocol in the hood?

All your materials are already inside, and no media or cells are opened until you are completely ready, so your hands stay in.

Can you open a culture flask outside the hood?

Never, not even at the microscope; the cells can be seen through the flask.

What is the glove rule?

Always wear gloves in the cell culture room.

Why are Bunsen burners banned this week?

People are spraying ethanol around the room, and nothing should be in flames.

How are your solution tubes marked and used?

With names and hood number in Fisher Brand markers that resist 70% ethanol, and you use only your own solutions.

Where do the solutions sit while you work in the hood?

Trypsin/EDTA and BCS in a small ice-filled insulated cup; DMEM+ and PBS at room temperature.

How are the solutions stored between labs?

DMEM+ and PBS at 4 degrees C, trypsin/EDTA and BCS at -20 degrees C, each in your section's rack.

Which waste containers go in the hood?

Two waste beakers, one for solids and one for liquids, with the 70% alcohol spray kept outside the hood.

What do you do while waiting for a hood?

Look at your cells on the inverted phase contrast microscope with the cap on, and photograph them with your phone.

Step 1: removing the old medium

Tilt the flask and pour the expired medium into the liquid waste beaker without disturbing the cells.

Step 2: the PBS rinse

Pipette 4 ml sterile PBS in at the neck, tilt to rinse the cells, then pour it out.

Step 3: adding trypsin

Add 1 ml trypsin/EDTA, tilt until it covers the whole surface, and check the cells every two minutes on the inverted microscope.

How can you tell the cells have detached?

Under the microscope they round up and float; tilt the flask until no attached cells remain.

Step 5: stopping the trypsin

Add 4 ml PBS and 0.5 ml BCS, then pipette the solution over the surface 3-4 times to rinse off any remaining cells.

Steps 6-7: collecting and spinning the cells

Pour the ~5 ml cell suspension into a 15 ml conical and spin at 1,200 rpm for 5 minutes (1,000 x g in the IEC centrifuge), as a group.

Step 8: after the spin

In the hood, pour off the entire supernatant, add 1 ml PBS, finger-vortex until the pellet has no clumps, and put the cells on ice.

Why finger-vortex the suspension right before seeding?

Cells left undisturbed for several minutes fall to the bottom of the tube.

How many cells go into the new flask, and in what?

6.6 x 10^4 cells with 4 ml of fresh DMEM+, swirled to spread them evenly.

How do you work out the volume of suspension to seed?

Divide 6.6 x 10^4 by the cells per ml of your suspension; the answer is the ml to add.

What happens to the new flask?

Label it with your name and put it in your section's basket; your TA moves it to the incubator.

What happens to the rest of the cell suspension?

It goes back to your bench in the 15 ml conical to be ethanol-fixed.

Where does solid waste go?

Into the red bag trash.

Where does the liquid waste go?

Into the 4 L brown glass culture waste jug near the window sink in room 184.

How is liquid culture waste treated?

A squirt of 10% bleach is swirled in the beaker and poured into the jug; used media must sit with bleach for at least 1 hour before going down the drain.

What is the last cleanup step for the hood?

Wipe down the hood surfaces with 70% alcohol.

Why can fixed cells be handled outside the hood?

Fixing kills the cells, so they will not be cultured again and need not stay sterile.

Which PBS is used for the fixation?

Non-sterile PBS, never your sterile PBS.

How are the cells spun and washed before fixing?

Spin at 1200 x g for 5 minutes, remove the supernatant with a P1000, and wash twice in 1 ml non-sterile PBS.

What are the cells resuspended in before the ethanol?

500 microliters of non-sterile PBS.

How is the ethanol added?

5 ml of ice-cold 70% ethanol, added drop by drop while vortexing.

Why vortex while the ethanol goes in?

Otherwise the cells are fixed to each other in clumps.

How is the ethanol kept cold?

Its tube stays in ice the whole time 1 ml volumes are being withdrawn.

What happens to the fixed cells?

They stay on ice in a firmly resealed tube labelled with initials, TA, lab room and day, and go to the TA for storage at -20 degrees C.

What are the fixed cells for?

Next week's propidium iodide (PI) staining and DNA content analysis; ethanol fixation lets the stain into the cells.

What is a hemocytometer?

A specialized slide that holds a thick cover glass on raised support strips, 0.1 mm above a platform etched with a 1 mm x 1 mm grid.

What volume lies above the central 1 mm square?

1 x 10^-4 cubic centimeters, that is, 1 x 10^-4 ml.

Formula for cells per ml

Cells counted in one 1 mm square x 10^4 = cells/ml.

How much suspension is loaded, and how is it viewed?

10-15 microliters, counted by phase contrast at 100X.

How is the chamber filled?

Put the filled pipette tip on the platform at the edge of the cover glass and release slowly until the space is filled; holding the cover glass with thumb and forefinger can help.

Rule for a cell lying on a grid line

Count it if it is on the top or left line, but not if it is on the bottom or right line.

How many cells must you count?

At least 200: count all cells in the central 1 mm2, and if there are fewer than 200, count a second 1 mm2 area of the same grid.

How is the hemocytometer cleaned and stored?

Rinse the cover glass and platform with dH2O, blot gently with a Kimwipe, store it face-down on a folded Kimwipe, and keep the cover glass in its plastic sleeve.

Practice: 250 cells counted in the central square. Cells per ml, and volume to seed?

250 x 10^4 = 2.5 x 10^6 cells/ml, and 6.6 x 10^4 / 2.5 x 10^6 = 0.026 ml, about 26 microliters (a worked example, my numbers).

JoVE: what is cell culture?

Growing eukaryotic cells in the lab, as a model system for studying cell physiology outside their natural environment.

JoVE: primary versus secondary cultures

Primary cultures grow straight from a tissue sample; cells subcultured from them into a new dish form secondary cultures.

JoVE: adherent versus suspension cultures

Most vertebrate cells grow attached as a monolayer on chemically treated ware; a few, such as hematopoietic cells, grow in suspension in untreated ware stirred by magnetic stirrers or spinner flasks.

JoVE: what do synthetic basal media such as DMEM and RPMI contain?

Amino acids, vitamins, inorganic salts and a carbon source such as glucose, supplemented with serum for growth and adhesion factors, hormones, lipids and minerals.

JoVE: how is the medium's pH held and shown?

About pH 7.4 for mammalian cells, held by a bicarbonate buffer with exogenous CO2, with phenol red to show pH changes.

JoVE: what is a plant explant culture?

Plant tissue grown on a nutrient medium containing plant growth hormones, micronutrients and a carbon source.

JoVE: what decides the tissue a plant culture forms?

The auxin to cytokinin ratio: a balanced ratio gives an undifferentiated callus, and changing it drives root or shoot formation.

JoVE: what are plant cell cultures used for?

Improved hybrid plants, conserving endangered plants and large-scale production of plant-derived products.