1
Q
What is the cellular response of a particular cell line when exposed to a new pharmaceutical?
A
- Expression
- Variability
- Localization
2
Q
What are Ptk2 cells?
A
- Mammalian cells from potoroo animals
- Risk group 1
- Adherent (due to ECM and attachment points)
- In our lab: glued themselves to the coverslip
- Infinite lifespan
3
Q
Term theme of lab:
A
How do Ptk2 cells respond to stress?
4
Q
What is apoptosis:
A
- PCD
- Can be due to stress
- Cell shrivels, cytoplasm shrinks, loss of adhesion, organelles fragment, cells dismantle (blebbing)
5
Q
What is necrosis?
A
- Cell death
- In response to severe trauma/stress
- Cell releases contents
6
Q
Lab 1 procedure:
A
Stress Ptk2 cells with vinegar (acid shock) or hydrogen peroxide (oxidative shock), compare morphology of stressed vs unstressed and estimate how many cells alive/dead. Unstressed cells are treated with vol of water that is identical to vol of vinegar/h2o2 in stressed cells
7
Q
What are primary cells?
A
- Taken directly from tissue
- Finite lifespan, may divide a few times but will eventually die—hard to grow
- Represent cells inside organism
8
Q
What are immortal cell lines?
A
- Divide indefinitely
- Originate as cancer cells or were modified in lab to get this ability
- Not good representation of cells inside organism, get further modified the more they divide
9
Q
Adherent vs suspension cells
A
Adherent cells: adhere to a substrate, form a solid monolayer (100% confluency)
Suspension cells: do not adhere to substrate
10
Q
Egs of immortal cell lines
A
- HeLa
- HEK293
- SF9
- Cancer cells: MCF7, Saos2, PC3
11
Q
Risk groups (4)
A
- RG1: Low risk eg Ptk2 cells
- RG2: Can cause serious disease but likely won’t eg SARS CoV-2-RNA, listeria, HEK293—all human cell lines at Dal are treated as RG2!
- RG3: Likely to cause serious disease eg SARS CoV-2 whole live virus, tuberculosis
- RG4: Can produce contagious/fatal disease there is no cure for eg Ebola
12
Q
Biological safety cabinet
A
- Important in CL2 labs
- Prevents aerosols from escaping and prevents unfiltered air from entering work area
- Air in cabinet is filtered thru HEPA filter
13
Q
Short term storage of growing cells
A
- Use CO2 incubator
- Provides clean and humidified environment at 37 celcius for mammal cells
- Have 5% CO2 which maintains pH
14
Q
Long term storage of growing cells
A
Cryopreservation in liquid nitrogen using DMSO to protect the cells from harsh conditions
15
Q
What type of microscope for cells growing on bottoms of flask? Why?
A
- Inverted microscope
- Have lens on bottom and light above specimen
- Good bc cells grow on bottom of flask and condensation is on the top of flask
16
Q
Components of commercially available media
A
- 5-10% fetal bovine serum (FBS)
- Antibiotics to minimize risk of bacterial growth
- Phenol red to indicate pH (acidic=orange, basic=purple)
17
Q
What do cell bio experiments test? How do we set this up?
A
Test effect of single variable on a measurable phenotype
- Grow cells
- Collect cells and determine their concentration
- Dilute them with media to conc of 1x10^5 cells/mL
- Seed them into flasks to begin experiment
18
Q
What is our variable?
A
Stress via acid or oxidative
19
Q
Containment level classifications (4)
A
- CL1: Regular teaching lab i.e. BIOL2020 lab
- CL2: Diagnostic/healthcare work, have BSCs i.e. tissue culture
- CL3: Need sealed windows and BSCs, i.e. covid lab
- CL4: Max level of containment, lab workers need full pressure suit and breathing supply i.e. national microbio lab
20
Q
Micropipette types
A
P20: 1-20ul
P200: 20-200ul
P1000: 200-1000ul
21
Q
When would you use a PipetBoy
A
For transferring volume larger than 1mL
22
Q
Microscope magnification calculations for light microscopy
A
10x: (10x)(10)= 100x
40x: (40x)(10)=400x
Uses ocular lens of 10x and objective lens of 10x or 40x
23
Q
Phase contrast microscopy
A
- Ideal to examine thin, living specimen
- Changes light intensity to help see blue colour of apoptotic cells
- 10x turret setting is Ph1, 40x turret setting is Ph2
24
Q
What is trypan blue?
A
- Stain used to determine cell viability (how many are alive and dead)
- Blue=dead, clear/gray=alive
25
Markers of apoptosis in microscope
- Membrane blebbing (small vesicles budding off)
- Cell shrinkage
26
What is a hemocytometer?
- Standard way of counting cells
- Slide with etched grid, count how many cells in each block
- Cells are usually loaded into hemocytometer with equal vol of trypan blue and squares
- We count the 4 big squares in the 4 corners
27
Equation to solve for cells per 0.1 ul
Cells per 0.1ul = number of cells counted/number of large squares containing counted cells
**Represents concentration of cells once they’ve been mixed with trypan blue
28
Equation to solve for cells per 1mL
Cells per mL= (number of cells counted/number of large squares containing counted cells) x dilution factor x 10^4
29
Equation for dilution factor
Dilution factor=(vol sample+vol diluent)/vol sample
30
Dilution formula
C1V1=C2V2
- C1: conc of live cells in the tube
- C2: mL of cells in tube initially
- C2: ideal final cell conc
- V2: how much media you need to add to tube to get C2
31
What does RIPA buffer do?
- Extracts proteins from cells by lysing cell membrane, collect supernatant AKA a cell lysate
- Lysate: liquid with a bunch of proteins
**used this in lab to get lysate of stressed cells and unstressed cells
32
What happens if lysate doesnt stay cold?
Proteins denature
33
Physical methods of getting lysate:
- Liquid homogenization: shears cell by forcing it thru narrow space
- Sonication: uses sound waves to probe and lyse cells
- Manual grinding: tissue is frozen in liquid nitrogen and then smashed w mortar and pestle to release proteins
34
Challenge with physical methods of getting lysate
- Can generate heat
- Less consistent, less reproducible
35
Chemical methods of getting lysate:
- Use detergents or hypotonic solutions to disrupt membrane
- Eg. RIPA—contains Triton X-100 detergent, as well as protease and phosphatase inhibitors
36
Process of using RIPA
- Grow cells in flasks
- Remove media, wash with buffer i.e. PBS
- Add RIPA buffer to flask
- Incubate cells in RIPA on ice for 10+ mins
- Transfer lysed cells onto tube and centrifuge contents—supernatant is cell lysate
37
How do we determine protein conc of cell lysate?
Bradford assay
38
What is a Bradford assay?
- When dye in Bradford reagent binds to protein, colour changes from brown to blue
- Intensity of blue is measured w spectrophotometer
39
What is BSA?
- Used in Bradford assays as the standard—shows signal so we can compare other proteins
- Bovine serum albumin
- Made of AAs
40
Preparing dilution series of stock BSA
- Dilute BSA with PIPES buffer
- Add 5mL Bradford reagent
C1V1=C2V2
C1: initial conc of BSA
V1: vol of BSA stock needed
C2: final BSA conc you want
V2: final vol total of solution you want
41
Preparing dilution series of lysates
- 95 ul of PIPES and 5 ul of cell lysate (either T or C)—this is a 20x dilution
- Add 5mL Bradford reagent
42
Spectrophotometer
- Absorbance of light by sample is measured and converted from light energy into electrical energy, so a reading is displayed
- Degree to which light is absorbed (AKA absorbance) is related to conc of absorbing solution
43
Absorbance results for dilution series of BSA
Higher BSA conc (AKA higher vol of stock BSA in solution) = higher absorbance
Solution with 0ul BSA will have 0 absorbance
44
Absorbance results for dilution series of lysates
Control lysates have higher absorbance than treatment lysates—meaning that stressed cells have a lower protein concentration
45
Solving for actual protein concentration of cell lysate from standard curve
y=ax formula given from graph
y is absorbance
x is protein conc in cuvette
a is slope of line
To solve for actual protein conc, solve for x and multiply by dilution factor
46
Graph preparation guidelines
- Caption below figure, no colon after number
- Caption includes: what variable is being measured and how, include eqn of line and R^2 value, no date/time/location, don't start with "Graph of"
- No gridlines or border, white background
- Key or legend if multiple plot lines
47
Table preparation guidelines
- Title above table, no colon after number
- Title includes: key info to understand table, no date/time/location, don't start with "table of"
- Thick line above and below table, thin lines below header, white background
- Indicate 2 decimal places for volumes
- Do not include column for “tube #” or “sample #” if it refers to numbered tubes used to set up the experiment
48
What is Hsp27?
A molecular chaperone that protects proteins, prevents misfolding, and regulates apoptotic pathways
** we want to know if stressed cells made more/less/no change of Hsp27
49
What is p53?
A tumour suppressor protein that monitors cell health (specifically DNA). If DNA is damaged, p53 halts the cell cycle so the cell has time to fix the damage. If it can’t be fixed, p53 initiates apoptosis
** we want to know if stressed cells made more/less/no change of p53
50
What is electrophoresis?
- Separates charged molecules in an electrical field
- Molecs are driven thru gel
- Large molecs move slower, small molecs move faster
- Neg charged molecs move further bc they are attracted to positive electrode
- PAGE (polyacrylamide gel electrophoresis)
51
What is SDS
- Sodium dodecyl sulfate
- Is used before SDS-PAGE
- Cell lysates are mixed with SDS sample buffer
- SDS disrupts protein folding, denaturing proteins
- Means that protein movement thru gel doesn’t depend on shape of protein
- Increases speed at which proteins migrate toward pos electrode (AKA will make all proteins neg charged)
52
What is B-mercaptoethanol and DTT
- Reducing agents
- In sample buffer with SDS to break disulfide bonds
53
SDS PAGE vs Native PAGE
SDS page uses SDS to denature proteins
Native page doesn’t denature proteins, can provide info abt protein structure/folding
54
What are molecular weight standards
Proteins with known molec weight are run thru gel alongside proteins, allowing us to compare migration distances to estimate molec weights of sample proteins
55
Calculations to prep cell lysate solution for SDS-PAGE
Need to run the same total number of ug of protein in each lane
Eg. 50ul of 1.0ug/ul solution of each cell lysate—use C1V1=C2V2 to calculate volume of lysate you will need
C1: conc of lysate
V1: how much cell lysate you need to remove from tube (unknown)
C2: final conc you want (1.0)
V2: final vol you want (50)
56
Why is heating and centrifuging important prior to SDS-PAGE?
Heating in hot water bath ensures proteins are fully denatured
Centrifuging removes insoluble material (anything that isnt the proteins we want
57
Settings of electrophoresis unit for SDS-PAGE
110mAmps and 200 volts
58
What is a turbo-blot machine?
After electrophoresis, gel is stacked onto nitrocellulose membrane and turbo-blot machine applies current to move neg charged proteins off gel onto nitrocellulose
59
What is Coomassie Blue?
- Can be used to stain gel in electrophoresis
- Allows us to see ALL proteins in cell lysate
- Not very useful, cant rly figure out which band corresponds to protein of interest
60
What are antibodies?
- Produced by animal immune sys
- Go against a target protein (antigen)
- Typically belong to IgG class, Y structure. Base of Y is constant and branches are variable
- Can be raised and collected from an animal host
61
2 general approaches to generate antibodies for research:
Polyclonal antibodies: Inject animal host with purified antigen/protein of interest. Antibodies animal makes are harvested, resulting in a mixture of antibodies each specific for a different epitope on antigen
Monoclonal antibodies: Inject animal host with antigen, triggers immune response. Antibody-producing WBCs are removed and fused with immortal cells that will continue to divide. They are specific for the same epitope on an antigen, and therefore are less likely to randomly bind to other proteins
62
What are reporter molecules?
Conjugated to antibody. May fluoresce under microscope or catalyze a rxn to produce colour change or light, letting us visualize antibody binding to antigen
63
Direct vs indirect visualization
Direct: Primary antibody is attached to reporter molec and binds to protein of interest
Indirect: Primary antibody binds to protein of interest, secondary antibody is attached to reporter molec and binds to primary antibody
64
What is HRP?
Horseradish peroxidase
Enzyme used in western blot
Catalyzes a rxn that releases light that we can see on film
Made of AAs
65
What info do we gain from HRP light in western blot?
Light intensity gives info abt expression of protein
Light location gives info abt molec weight of protein
66
Purpose of western blot:
Determine protein expression levels of target protein
67
Importance of procedural check in western blot
- Use housekeeping proteins with constant and predictable expression
- We wouldnt expect these proteins to up or down regulate in our conditions
- Ensures that there were no issues with our procedure
- Eg of proteins used for check: tubulin or actin
- Will look the same in both treatment and control
68
What is Ponceau Red used for?
- To confirm lysates transferred to nitrocellulose, allows us to see and label lanes
- Stains membrane red, ensures we can see pre-stained marker bands and cell lysate proteins
- Is temporary
69
Importance of milk protein
- Used in western blot
- Called casein
- Covers nitrocellulose w milk protein
- Reduces nonspecific binding of antibody to nitrocellulose membrane and other proteins than the one we are interested in
70
Importance of TBS-tween
- Used in western blot
- Poured onto nitrocellulose after it has incubated in antibody
- Washes away unbound antibody
71
Importance of TBS buffer
- Used after TBS-Tween in western blot
- Tween interferes with chemiluminescence, ensures Tween is fully gone
72
How long should nitrocellulose sit in antibody
- Used 15 mins in our lab
- Longer it sits, more time proteins have to bind to antibodies efficiently
73
What does luminol/oxidizing reagent mixture do
When it is put onto nitrocellulose after conj to antibody and rinses with Tween and buffer, the HRP on the antibody catalyzes oxidation of luminol, causing light release (black lines) where our protein of interest is
74
How to analyze a Western blot
- Using blue autoradiography film in a dark room
- Light signal causes black lines on film
1. Compare to nitrocellulose—are proteins detected at expected molec weights we probed for?
2. Ensure procedural checks went smoothly
3. Look at band intensity (thickness) of control (unstressed) to see baseline expression, and compare to treated (stressed) sample to see if protein expression was up- or down-regulated
75
Why would control have no lines?
1. Membrane doesnt spend enough time in anti-tubulin/actin antibody and antibody may not have bound to tubulin
2. Cell lysates had low tubulin
76
Should stress cells have up- or down-regulated Hsp27 and p53?
Up-regulated, as they pause cell cycle/induce apoptosis…stressed cells should interact with them bc the cells are damaged
77
Unit of measure for molec weight
kDa
78
Antibody naming conventions
Direct:
Made by rat, recognizes bloop protein, reporter attached is FITC
= Rat anti-bloop, labelled with FITC
Primary indirect:
Made by rat, recognizes bloop protein
= Rat anti-bloop
Secondary indirect:
Primary is injected into goat, reporter attached is FITC
= Goat anti-rat, labelled with FITC
79
What are fluorochromes
They absorb photon and then emit light at a higher wavelength which produces colour
80
What is FITC?
- Fluorochrome that emits green light
- Accepts photon at 495nm and emits at 517nm
- Is a reporter molecule, antibody can be conjugated to it
81
What is Hoescht?
- Can label DNA
- Is a fluorochrome
- Never conjugated to an antibody
- Emits blue light at 460-490nm
82
What is the importance of methanol
- Fixes cells so their structures stay intact (kills them)
- Permeablizes cell membrane so antibodies can enter the cell and bind to target protein
- Works better being ice-cold
83
Why is Hoescht dangerous?
Is a potential mutagen and carcinogen bc it binds DNA. Should wear gloves and be cautious using it
84
Alternative fluorescence probes
- Eg. GFP
- Allows us to detect protein localization in alive, unfixed cells
- GFP glows green
85
Confocal microscopy
- Centers light through a pinhole to focus on one plane
- Gives a more high quality image than traditional microscopy
86
FRET
- Fluorescence resonance energy transfer
- If proteins move close to eachother, energy from one fluorochrome is transferred to the other
- Used to measure interaction and distance between proteins
87
Flow cytometry
- Gains info abt a bunch of cells
- Laser scans cells and computer generates info abt shape and size of cells
88
FACS
- Fluorescence-activated cell sorting
- Variation of flow cytometry
- Cells are separated into droplets, can physically separate cells for future use based off specific traits
89
How to present microscopy images
Figure caption below image, including:
Specimen, what protein/genetic material each colour is referring to, microscopy type, total magnification, any imaging software used
90
How did we visualize DNA and tubulin in lab
FITC binds to tubulin in our lab, and Hoescht binds to DNA. We use UV filter on fluorescence microscope to see blue DNA and green microtubules
91
First steps of collecting cells
1. Remove old media. Adherent cells attached to bottom of flask in old media
2. Cells stay stuck, so wash with PBS
3. Add trypsin to lift cells
4. Add fresh media for cells to float in
5. Now you can split, count, or use cells for experiment
92
What does PBS do
- Phosphate buffered saline
- Removes residual Ca2+ and FBS from any leftover media
- Ensures that when we add trypsin, the enzyme will remain active to lift cells from vessel wall
93
What must happen before you can manipulate adherent cell cultures (move them, count them, set up exp, extract proteins, etc)?
- Cells must be detached from bottom of culture vessel and collected using proteolytic enzyme trypsin
94
SDS sample buffer components
SDS, B-mercaptoethanol, DTT, glycerol, tracking dye
95
Tracking dye in SDS sample buffer
- Allows us to follow progress of electrophoresis, stopping when it reaches the bottom
- Charged blue dye migrates thru the gel ahead of the smallest proteins
96
Glycerol in SDS sample buffer
Helps make samples sink into bottom of well
97
What is chemiluminescence?
- HRP on the antibody will catalyze oxidation of luminol, causing light to be released. The glow is not intense enough to be detected by the naked eye, so alternative methods are used
- Automated systems read the light exposure and generate an image (we use blue autoradiography film) and in a dark room, light signal from HRP is captured on film-resulting in black lines on the blue film.
- Any location on the film that was exposed to light will turn black (more light intensity results in a darker and thicker band)
98
What is Cy3.5?
- Cyanine 3.5
- Emits red light
- Absorbs photon at 579nm, emits at 591nm
99
What does anti-fade mounting medium do?
- Glycerol-based mountant applied to slide, and then coverslip is placed ontop cell-side down
- It is applied directly onto fluorescently labelled cells to preserve their fluorescence
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