1
Q
What did we look at all semester?
A
This semester we will look at thermoregulation in mammalina cells.
- We know that animal cells grow best at 37 degrees, but what happens if cultured cells are exposed to jhigher or lower temperatures
2
Q
What are we doing in lab 1?
A
We are looking at an extreme case - exposing mammalian cells to freezing temperatures in the prescence or absence of a cyroprotective agent.
- We will observe cell viability after 40 minutes of freezing.
3
Q
When should we take off our lab coats?
A
Lab coats should be removed prior to leaving the lab and they should never be worn outside to prevent any contamination spreading outside of the lab.
4
Q
When do you use double gloves?
A
If you have an open sore on a hand, you should put on a bandage and use double gloves.
5
Q
What are the learning outcomes of this lab?
A
- demonstrate pipetting proficiency
- describe how mammalian cells are maintainedd in vitro
- collect adherent cells growing in flasks
- explain differences in brightfield and phase contrast
- calculate cell concentration using a hemocytometer
6
Q
What is done during lab one?
A
- practice micropipetting
- conduct a simple experiment on cyroprotective agents using mammalian cells
- examine cells using brightfieqld and phase contrast microscopy
7
Q
What are the big themes for this term?
A
Thermoregulation
Call Viability
Protein Expression
8
Q
How will we examine thermoregulation?
A
What happens to cells when they are stressed by temperature changes
9
Q
How will we examine cell viability?
A
How do you count cells and how can you tell if they are dead or alive
10
Q
How will we examine protein expression?
A
How can we tell is cells are making more or less of a particular protein
11
Q
What is the most accurate way to transfer small amounts of liquid?
A
The most accurate way to transfer small volumes of liquid is to use a micropipetter.
12
Q
What are the three micropipettes that we use?
A
We will be using a P20, P200 and P1000 throughout the lab.
13
Q
Each typre of pipette is specially designed for what?
A
Each type of pipette is optimized to dispense a specific range of volume. You will know which one is which by reading the label at the top of the plunger.
14
Q
When do we use the P20, P200, and P1000?
A
P20: 1-20ul
P200: 20-200ul
P1000: 200-1000ul
15
Q
P20 black and red notches?
A
black indicates 1-20ul
red # at bottom indicates tenths (0.1)
red at bottom indicates hundredths (0.01)
16
Q
P200 black notches?
A
All number are black:
top is 20-200
bottom is tenths (first decimal)
17
Q
When putting liquid into the pipette wgat do we do?
A
Push the plunger to the FIRST stop
- put tip in solution and smoothly and gently let go of the plunger
- slowly push plunger pack down when at a new tube
- push to second stop to expel all liquid
- only release tip when all liquid is gone and the tip is removed from soluton
18
Q
P1000 black and red notches?
A
red = top position (0 unless 1000ul)
black # indicate 200-999
black notch = individual microlitres
19
Q
Bubbles?
A
Bubbles are bad in micro pipetting - bubbles and froth take the place of liquid, meaning that we do not transfer the volume that we think we are.
20
Q
What do we use when we need to transfer a larger volume than 1ml (1000ul)
A
We use a pipet boy.
TOp button = sucks liquid in
Bottom button = releases liquid
21
Q
Things to avoid in transferring large amounts of liquid?
A
- avoid touching the part of the pipette that will be in contact with the solutions
- avoid bumping the pipette on any surface
- push on the upper bottom to draw up any liquid
- DO NOT suck liquid into the pipetboy
- push lower button to expel liquid
- dispose of the pipette (manually pull off te pipette)
- bubbles are bad
22
Q
What is the important part of growing cells in vitro?
A
The ability to grow cells in vitro is a fundamental part of molecular biology research.
23
Q
Growing cells in lab can also be used for what purpose?
A
Growing cells in the lab can also be used for clinical purposes (diagnostics, personalized medicine) and for practical biotechnology (production of proteins, pharmaceuticals, and viruses)
24
Q
What are primary cells?
A
Primary cells are taken directly from a tissue. Unless modified in some way, these cells have a finite lifespan. They may divide a few times but will eventually die.
25
WHat are primary cells useful for?
Primary cells are excellent for some applications because they closely resemble in vivo physiology (i.e. they are more representative of the cells in the organism), but they have the disadvantage of being a bit trickier to grow, and ultimately, dying.
26
What are immortal cell lines?
Immortal (‘Continuous’) cell lines divide indefinitely. These cells may have originated as cancerous cells, or they may have been transformed in the lab to give them this ability.
27
What are advantages and disadvantages of immortal cell lines?
The advantages of immortalized cells are that they are easier to grow and that they divide over and over. However, they are less representative of in vivo systems, and over time will acquire increased levels of genetic modifications.
28
What two forms can cell lines take?
Cell lines can we adherent or grow in suspension
29
What do adherent cells do?
Adherent cells attach to a substrate and cells growing in suspension do not. Adherent cells will eventually grow and divide to the point where they form a solid monolayer,
with minimal space between the cells.
- This is referred to as 100% confluency. Depending on the particular cell line, some cells will stop growing at this point, while other types of cells (like cancer cells) may continue growing on top of each other.
30
What are some common immortal cell lines used in labs around the world?
- HeLa
- HEK 293
- SF9
- MCF-7
- Saos-2
- PC3
31
What is HeLa?
This is the first human immortal cell line. It was established in 1951 and was derived from a cervical cancer tumour.
32
What is HEK 293?
This is a human embryonic kidney epithelial cell
33
What is SF9?
These are insect cells that can be grown in solution.
34
What are other examples of cancer cells?
MCF-7: breast cancer
Saos: bone cancer
PC3: prostate cancer
35
What is biosafety? In Canada, what is biosafety regulation oversight done by?
Biosafety is the use of practices, equipment, and facilities to prevent personal, laboratory, or environmental exposure to biohazardous materials.
- In Canada, biosafety regulation oversight is by both federal (PHAC and CFIA) and provincial (OHSA)
36
What molecules are assigned risk groups? What are the classifications determined by?
All microorganisms, proteins, and nucleic acids are assessed to determine their risk to the individual/animal and public health.
- They are assigned to one of four risk group (RG) categories.
- These risk group classifications are
determined by the Public Health Agency of Canada and the Canadian Food Inspection Agency.
37
Is included with risk group 1?
Very low risk. e.g. Saccharomyces cerevisiae (yeast), CHO cells, non-pathogenic E.coli.
38
What is in risk group 2?
These pathogens are capable of causing serious disease in a human but are unlikely to do so.
- For example, Listeria monocytogenes, HEK293, Norwalk virus, SARS-CoV-2 RNA. At Dalhousie, all human cell lines are treated as Risk Group 2.
39
What is in risk group 3?
These pathogens are likely to cause serious disease in a human or an animal, but effective treatments are available. e.g. Mycobacterium tuberculosis, SARS-CoV-2 whole live virus.
40
What is in risk group 4?
These pathogens can produce highly contagious, serious or fatal diseases for which there are no treatments or vaccines. e.g. Ebola virus
41
What does containment level refer to?
Containment levels refer to the minimum physical containment and operational practices required for safe handling of infectious materials and toxins. There are four containment level (CL) classifications
42
What is in CL1?
This is a “regular” type of teaching lab. They do not have any special design features other than a
functional working space and cleanable work surfaces. Open bench work is acceptable and Biological
Safety Cabinets are not required. Our regular lab rooms (LSC 7009 and 7012) are CL1 workspaces
43
What is in CL2?
This is a common type of facility in hospitals and universities for either diagnostic, health-care work or for
research purposes. Generally, all RG2 pathogens are contained in CL2 facilities. These rooms have BSCs
that are equipped with HEPA filters. The tissue culture rooms in the LSC are CL2
44
What is CL3?
These laboratories require additional primary and secondary barriers to minimize the release of infectious
organisms into the environment. This includes sealed windows, the use of a BSC for all work and strictly controlled access. Canada has over one hundred CL3 laboratories
45
What is CL4?
These facilities provide the maximum level of biosafety and biosecurity. Maximum containment is ensured
via a complete seal of the facility perimeter, which includes sealing any conduits crossing the containment
barrier, such as electrical and plumbing. The lab worker must wear a full coverage positive-pressure suit
with its own breathing supply. There are only two CL4 laboratories in Canada; the National Microbiology
Laboratory, and the Canadian Science Centre for Human and Animal Health, both in Winnipeg.
46
What cells are we using this term?
We are using CHO (Chinese hamster ovary) cells.
47
What are CHO cells derived from, and what type of cell line are they?
This term we will use Chinese Hamster Ovary (CHO) cells. As the name implies, these are cell line derived from a biopsy of an ovary of an adult, female Chinese hamster. This is an immortal cell line that has been dividing in culture since 1957! These cells are commonly used around the world in biotechnology research.
48
What is the risk group classification of CHO cells, and why can we use them?
Because these cells are RG1, we can
work with them in our teaching labs (CL1 workspaces).
49
What is a Biological safety cabinet?
A Biological Safety Cabinet (BSC), i.e. the “hood”, is the single most critical piece of equipment needed for working in a CL2 lab.
- A BSC protects you from infectious materials or toxins AND protects your specimens from contamination.
50
BSC compared to fume hood?
although superficially they may look very similar, a BSC is NOT a fume hood (which simply sucks the air from the hood out a vent, without filtering)
51
What does a BSC do when it is properly functioning?
A properly functioning BSC creates an air curtain across the front opening.
- This prevents aerosols from escaping out the front AND prevents unfiltered air from entering the working area. This air curtain is fragile, so care must be taken to not disturb it.
- Inside the hood, the air moves in a
constant, streamlined speed and direction, which creates a laminar flow that contains airborne
infectious agents.
- The air from the cabinet is exhausted through a HEPA (High Efficiency Particulate Air) filter. BSCs also maintain a sterile work environment by filtering the incoming air through a HEPA filter before it blows across the working surface.
52
What is a CO2 incubator required for?
A CO2 Incubator is required for short-term storage of growing cells.
53
What do CO2 incubators provide and what are they supplied with?
The incubator provides a clean, humidified environment with a constant temperature (usually 37 ̊C for mammalian cells). The incubators are supplied with 5% CO2 which maintains the pH at physiological level.
54
What is different about inverted microscopes?
Inverted microscopes have the lens on the bottom of the microscope and the light source above the specimen.
- This type of microscope is necessary as cells are usually growing on the bottom of the flask and there is often condensation on the top of the flask.
- Additionally, flasks and dishes are bulky, and the large stage provides an adequate space to view the vessel.
55
What is commercially available media used for?
Commercially available media is used to provide the nutrients that the cells require. Media typically contains about 5-10% fetal bovine serum (FBS).
56
What does FBS provide?
FBS provides growth factors,
hormones, nutrients and electrolytes that eukaryotic cells need to survive. It is present in media in about 5-10%.
57
What are antibiotics? Why are they added?
Antibiotics such as penicillin and streptomycin are often added to the media to minimize the risk of bacterial growth following contamination.
58
What is phenol red? What do different colours of it indicate?
The media is pink due to the presence of the pH indicator
Phenol Red.
- If the pH becomes acidic the media will turn orange, if the pH becomes basic it will turn purple.
59
What does a pH change indicate?
A change in the pH indicator colour is a sign that there is an excess of metabolic by-products and that it is time to either split the cells (also referred to as “passaging” or
“subculturing”) if they are confluent or change the media to replenish depleted nutrients.
- A change in pH can also indicated that your culture might be contaminated with bacteria, yeast, or fungi
60
Where are mammalian cells grown?
Mammalian cells are grown in specialized culture vessels that
have been treated to allow adherent cells to attach to the bottom surface. Examples include flasks, which have screw tops that can be either vented or non-vented, and plates, which
have individual wells.
61
What do we need when we want to move cels from one vessel to another?
When a lab technician wants to move an adherent cell line from one vessel to another, it is necessary to dislodge the cells from the vessel walls using a protease, called trypsin.
62
What does trypsin do?
This enzyme will cleave the proteins present in the cellular attachment points. When those proteins
are digested, the cells will lift off of the vessel wall and float in the liquid.
63
DMSO use in long term storage?
For long term storage, cells are frozen in media mixed with DMSO – a special cryopreservation agent that reduces the formation of ice crystals.
64
Where are cells initially cooled and where are they transferred?
The cells are initially frozen using a special container filled with isopropyl alcohol that will cool the samples at a rate of cooling at 1°C/minute (the optimal rate for cell preservation).
- In the BIOL 2020 labs, we use a container called a Mr. Frosty™ Freezing Container.
- Once cells are fully frozen, they are transferred to a liquid nitrogen Dewar for long-term storage below -130°C.
65
What occurred 24 hours before lab #1?
Approximately 24 hr prior to your lab time, we seeded (i.e. “added”) CHO cells into flasks.
- By the time you arrive in the lab, the cells should be well adhered to the bottom surface of the dish and have grown to our desired confluency. Each bench (of ~4 students) will
receive one dish containing cells.
66
What are the initial wash steps for the CHO cells?
1. Wear gloves.
2. Use PipetBoy + 10 ml pipette to remove old media. WARNING: Do not suck liquid into PipetBoy. Dispose in waste beaker.
3. Use P1000 to add 1 ml PBS.
4. Swirl over cells for ~5 seconds.
5. Remove PBS with same tip. Dispose tip in yellow Sharps container.
67
How do you perform the Trypsinization (detaching) step?
1. Add 500 ?l trypsin (P1000).
2. Rotate flask to coat cells; tap side of vessel gently.
3. Wait ~3 mins.
4. Check in Room 7011 on inverted scope (cells must be floating). Tap again if stuck.
68
How should microcentrifuge tubes be prepared while waiting for trypsin?
1. Prepare microcentrifuge tubes.
2. Label lids with initials.
3. Pair 1: Label side 'Media'.
4. Pair 2: Label side 'DMSO'.
(Note: Groups of 3 prepare both samples).
69
How do you neutralize the trypsin and split the cells?
1. Once cells float, add 500 ?l fresh media to flask.
2. Pipette up and down gently to mix.
3. Use P1000 to transfer 500 ?l of cells into each prepared tube (equal volumes).
70
What is the centrifugation procedure for the cell split?
1. Spin tubes in mini centrifuge for 30 seconds.
2. Check for cell pellet at bottom.
3. Use P1000 to remove supernatant into waste beaker.
4. WARNING: Do not disturb the pellet.
71
How do you resuspend the cell pellets for the Media vs DMSO tubes?
Pair 1 ('Media' tube):
1. Add 500 ?l fresh media to pellet.
2. Resuspend with pipette.
Pair 2 ('DMSO' tube):
1. WEAR GLOVES (DMSO is toxic).
2. Add 500 ?l media + DMSO to pellet.
3. Resuspend with pipette.
72
What are the storage and cleanup steps after resuspension?
1. Place tubes in -20 freezer rack.
2. Set timer for 40 mins.
3. Dispose flask and pipettes (5 & 10 ml) in biohazard bag.
4. Dispose tips in Sharps container.
5. Return solutions (PBS, Trypsin, media, DMSO) to fridge.
6. Dispose gloves and wash hands.
73
What is the principle and interpretation of the Trypan Blue viability test?
1. Principle: 'Exclusion test' (intact membranes exclude dye).
2. Interpretation:
- Clear/Gray cells = Alive (Intact membrane).
- Blue cells = Dead (Compromised membrane allows dye in).
74
What is the standard procedure for counting cells on a hemocytometer?
1. Use a hemocytometer (slide with etched grid).
2. Pipette cells into the chamber on top of the grid.
3. Counting Area: Count the cells in the 4 corner squares (A, B, C, and D).
75
How do you calculate the Dilution Factor for cell counting?
Formula:
Dilution Factor = (Volume Sample + Volume Diluent) / Volume Sample
76
What is the formula for calculating the final concentration (Cells per ml)?
Cells per ml = (Cells Counted / Squares Counted) x Dilution Factor x 10^4
77
How do you calculate total magnification on the Olympus CX41?
Formula: Total Magnification = Ocular Lens (10x) x Objective Lens.
- At 10x Objective: (10x)(10x) = 100X Total.
- At 40x Objective: (10x)(40x) = 400X Total.
78
What is Kohler Illumination?
1. Definition: Aligning the light path (bulb -> field iris -> condenser iris -> eye).
2. Note: This has been done for you. Ensure you do not see shadows or dark spots.
79
How do you set up the microscope for Brightfield Microscopy?
1. Use for: Specimens with natural color or stained specimens.
2. Procedure: Set turret position to 'O'.
80
How do you set up the microscope for Phase Contrast Microscopy?
1. Use for: Unstained, living, transparent/thin specimens.
2. Procedure: Align phase plate with turret filter.
3. Settings (Critical):
- If using 10x Objective -> Set Turret to 'Ph1'
- If using 40x Objective -> Set Turret to 'Ph2'
81
How do you prepare the microscope for the viability test (Hands-on Task #3)?
1. Plug in and turn on.
2. Set to 10x on Brightfield (Check turret).
3. Focus on Corn Stem slide (Bring stage ALL THE WAY UP, then slowly lower).
4. Switch to Phase Contrast. Adjust light intensity.
5. Leave setup ready for cells.
82
How do you prepare the cell sample for the hemocytometer (Hands-on Task #3)?
1. Retrieve cells from freezer (after 40 min).
2. In a NEW tube: Mix 20 ?l cells + 20 ?l Trypan Blue.
3. Pipette up and down to mix.
4. Place coverslip on hemocytometer.
5. Load 10 ?l of mixture into chamber.
83
How do you perform the observation and estimation for Task #3?
1. Place hemocytometer on stage.
2. Find grid using Phase Contrast (10x).
3. Switch to Brightfield (Turret 'O', lower light) to distinguish Blue (dead) cells.
4. Switch back and forth to visualize best.
5. Estimate % living vs dead.
6. Record observation.
84
How do you clean the hemocytometer?
1. Remove coverslip -> Dispose in yellow Sharps container.
2. Rinse chamber with 70% ethanol.
3. Wipe with KimWipe.
4. Return to box.
85
What is the final cleanup checklist for the microscope and bench?
1. Microscope: Ensure 10x objective is in place.
2. Microscope: Ensure turret set to 'O'.
3. Microscope: Turn off, unplug, apply dust cover.
4. Bench: Spritz with 70% ethanol and wipe down.
5. Personal: Wash hands with soap and water.
BIOL2020 Lab
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