Exam: Lab Content (Notes from Lab Manuals) Flashcards
Explain Lab 1: Cell Culture.
- stress Ptk2 cells using either vinegar (acid shock) or hydrogen peroxide (oxidative stress).
- also have a flask of unstressed cells.
- after 30 minutes, cell morphology was compared between stressed and unstressed cells and we estimated how many cells in each culture are living or dead.
What are primary cells?
- primary cells are directly from a tissue.
- have finite lifespan (may divide a few times but will eventually die)
- closely resemble in vivo physiology
- disadvantage of being a bit trickier to grow, and ultimately, dying
What are immortal cell lines?
- divide indefinitely
- originated as cancerous cells, or they were transformed in the lab to give them this ability.
- easier to grow and that they divide over and over
- less representative of in vivo systems, and with time acquire increased levels of genetic modifications
Compare adherent or suspension in terms of cell lines?
- 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 (100% confluency)
Containment levels refer to the minimum physical containment and operational practices required for safe
handling of infectious materials and toxins. Name and describe the use of all four.
CL1
- This is a “regular” type of teaching lab.
- Open bench work is acceptable and Biological Safety Cabinets are not required
CL2
- hospitals and universities for either diagnostic, health-care work or for
research purposes.
- RG2 pathogens are contained in CL2 facilities
CL3
- require additional primary and secondary barriers to minimize the release of infectious organisms into the environment.
- sealed windows, the use of a BSC for all work and strictly controlled access
CL4
- These facilities provide the maximum level of biosafety and biosecurity.
- complete seal of the facility perimeter, sealing any conduits crossing the containment barrier, electrical conduits and plumbing.
- lab worker must wear a full coverage positive-pressure suit with its own breathing supply.
What is a Biological Safety Cabinet (BSC), i.e. the “hood”? What does this do?
- needed for workingin a CL2 lab.
- protects you from infectious materials or toxins AND protects your specimens from contamination.
- When the researcher starts working, they slowly and deliberately put their hands into the hood, coming in from the front, and then they wait a few seconds to allow the air curtain to be re-established
- the air moves in a constant, streamlined speed and direction, (laminar flow) that contains airborne infectious agents.
- air from the cabinet is exhausted through a HEPA (High Efficiency
Particulate Air) filter. - maintain a sterile work environment by filtering the incoming air through a HEPA filter before it blows across the working surface
What is a CO2 Incubator?
- CO2 Incubator is required for short-term storage of growing cells.
- provides a clean, humidified environment with a constant
temperature (usually 37 ̊C for mammalian cells!) - incubators are supplied with 5% CO2 which maintains the pH at physiological level.
- long term storage, cells are cryopreserved in liquid nitrogen.
Inverted microscopes
- have the lens on the bottom of the microscope and the light source
above the specimen. - necessary as cells are usually growing on the bottom of the flask and there is often condensation on the top of the flask.
What is Media? What do you add to it to minimize bacterial growth? Why is it pink?
- media is used to provide the nutrients that the cells require & contains about 5-10% fetal bovine serum (FBS).
- Antibiotics (penicillin and
streptomycin) are often added to the media to minimize the risk of bacterial growth following contamination. - The media is pink due to the presence of the pH indicator Phenol Red
Phenol Red
pH indicator
- pH becomes acidic the media will turn orange; if the pH becomes basic it will turn purple
- 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
How 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.
Describe Ptk2 Cell Line
- marsupial cell line (CL1 LAB) = Ptk2 cells
- epithelial kidney cells from a male potoroo
- only a small number of chromosome
- Ptk2 cells stayed relatively flat in culture, making it quite easy to
see their large chromosomes. - these characteristics made this cell line ideal for studying genetics and the cell cycle
How do you set up experiments using cell cultures?
Many experiments in cell biology test the effect of a single variable on some sort of measurable phenotype.
We set up these types of experiments by:
1. Growing large quantities of cells.
2. Collecting the cells in a tube and determining the concentration of those cells.
3. Diluting the collected cells with media to a final concentration of 1 x 10 5 cells/ml (which is a commonly
used seeding concentration.)
4. Once the cells are diluted, they are seeded into flasks or plates to start an experiment
Trypan Blue
- is a common stain that is used to determine cell viability (i.e. how many cells are alive and how many
are dead?) - Dead, or dying cells with compromised cell membranes will allow the blue dye to leak into the
intracellular space
Blue = dead
Grey/clear = alive
Hemocytometer
- used to count cells
- a specialized slide with an etched
grid. - cells are pipetted into a chamber on top of the grid, which makes it easy to count how many cells are in each block
How do you determine cell concentration? What is the equation?
cells per ml = # cells counted / # of large squares containing counted cells
x dilution factor x 10^4
dilution factor = volume sample + volume diluent / volume sample
Using light microscopy, compare membrane blebbing vs cell shrinkage.
Membrane blebbing
- Once apoptosis signaling pathways have been initiated, the cell will start to
fragment and small vesicles will bud off from the membrane.
- This is one of the most defining
characteristics of apoptotic cells.
Cell shrinkage.
- As the cell fragments, it will become smaller in size.
- necrotic cells will swell and appear larger than normal.
Phase Contrast Microscopy
Phase Contrast Microscopy
- phase contrast makes highly transparent objects more visible by converting differences in light phase shifts into differences in light intensity.
- Phase contrast is ideal for examining thin, living specimens.
Cell seeding
Cell seeding is usually the first protocol step and a standard procedure in cell-based experiments. A correct and standardized cell seeding protocol is a critical factor for reproducible experimental results. The main challenge in this step is to achieve and maintain comparable cell numbers in all repeated experiments.
Trypsin
Trypsin is used to cleave proteins holding the cultured cells to the dish, so that the cells can be removed from the plates.
Procedure to collect cells
There are two cell culture techniques to grow cells in culture, as monolayers on an artificial substrate (i.e., adherent culture) or free-floating in culture medium (suspension culture).
Explain Lab 2: Protein Extraction and Quantification
You will receive tubes of Ptk2 cell lysate proteins that Mindy and Laura prepared during the summer. Your task is to determine the total protein concentration in each sample by performing a Bradford Assay.
Goal:
- Labs 2 through 4 are all about processing your cell lysate samples to compare the expression of a
particular protein (either Hsp27 or p53) in your control and treated samples
What is the Bradford Assay generally?
- The assay is simple; when a dye in the Bradford reagent binds to protein, there is a colour change from brown to blue.
- The intensity of the blue colour is measured using a spectrophotometer set at 595 nm.
- This assay uses BSA (bovine serum albumin, iolsated from cow blood) as a
standard to which the unknown samples will be compared
Describe an overview of the Bradford Assay used in Lab 2, highlight dilution.
- You will start by making a dilution series from stock BSA, which is at a concentration of 1.4 μg/μl.
- The colour change is linear only in the
range of 0.20 – 1.0 μg/μl, therefore the BSA must be diluted with PIPES buffer to make a series of standards in this range. - You will prepare 100 μl of each concentration.
- You will also set up cuvettes for the cell lysates (two cuvettes for the stressed cell sample (“T”) and two cuvettes for the unstressed cell sample (“C”). These samples will also be diluted
with buffer.
RIPA
- lysis buffer that contains the detergent Triton X-100
- typically includes protease and phosphatase inhibitors
How do you lyse cells using RIPA creating a cell lysate?
- Grow the cells in flasks.
- Remove the media and wash the cells with buffer (like PBS) to remove residual media.
- Add RIPA buffer to the flask, ensuring all cells come in contact with the buffer.
- Incubate the cells in RIPA buffer on ice for 10+ minutes.
- Transfer the lysed cells to a tube on ice and centrifuge the contents to pellet any membranes. The supernatant will contain a “soup” of the cell proteins. We refer to this as the cell lysate.
