LAB TEST Flashcards
What are the 3 things we looked at throughout the labs?
- Morphology - what does the entire cell look like?
- Protein expression - what happens to the expression of a particular protein?
- Protein localization - what happens to the location of a particular protein?
Lab 1 guide
Primary cells
- taken directly from a tissue
- unless modified, have a finite life span => may divide a few times but eventually die
- closely resemble in vivo physiology (advantage)
- trickier to grow, ultimately die (disadvantage)
Lab 1 guide
Immortal (‘continuous’) cell lines
- divide indefinitely
- originated as cancer cells or were transformed in lab to give them this ability
- easier to grow and divide over and over (advantage)
- less representative of in vivo systems and, with time, require increased levels of genetic modifications
Lab 1 guide
Adherent vs suspension cell lines
adherent
- attach to a substrate
- grow and divide to produce a solid monolayer, with minimal space between cells (100% confluency)
- some cells stop growing at this point, others (like cancer cells) continue growing on top of each other (depends on cell line)
substrate
- do not attach to a substrate
Lab 1 guide
Common immortal cell lines used in labs
- HeLa => first human immortal cell line, derived from cervical cancer tumor
- HEK 293 => human embryonic kidney epithelial cells
- SF9 => insect cells that can be grown in suspension
- MCF-7, Saos-2, PC3 => cancer cells
Lab 1 guide
Risk Group (RG) classifications
All microorganisms, proteins, and nucleic acids are assessed to determine their risk to the individual/ animal and public health
RG 1: very low risk (e.g., saccharomyces cerevisiae, Ptk2 cells
RG 2: pathogens are capable of causing serious disease in human but are unlikely to do so (e.g., listeria, HEK293, Norwalk virus, Sars-CoV-2 RNA)
RG 3: pathogens are likely to cause serious disease in humans or animals (e.g., mycobacterium tuberculosis, SARS-CoV-2 whole live virus)
RG 4: pathogens can produce highly contagious, serious or fatal disease for which there are no treatments or vaccines (e.g., ebola virus)
Lab 1 guide
Containment Facilities
Containment levels refer to the minimum physical containment and operational practices required for safe handling of infectious materials and toxins
CL1: ‘regular’ type of teaching lab, no 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 (Us!)
CL2: common type of facility in hospitals and universities for either diagnostic, health-care work or for research purposes. All RG2 pathogens are contained here. Rooms have BSCs equipped with HEPA filters
CL3: require additional primary and secondary barriers to minimize the release of infectious organisms into the environment, including sealed windows, use of BSCs for all work, and strictly controlled access (used for COVID research)
CL4: provide max level of biosafety and biosecurity. Max containment is ensured via a complete seal of the facility perimeter (includes sealing any conduits crossing the containment barrier, like electrical conduits and plumbing). Lab workers must wear full coverage, positive-pressure suits with their own breathing supply. Must go through a chemical shower before removing protective clothing
Lab 1 guide
Personal Protective Equipment (PPE) in the lab
- Lab coats - also needed to not track contaminates out of lab
- Proper footwear
- Gloves
Lab 1 guide
Ptk2 cell line
- epithelial kidney cells from a male potoroo (marsupial)
- isolated cells in 1962 were placed in culture dishes where they continued to replicate
Lab 1 guide
Potoroo (why use their cells??)
- they have a small number of chromosomes
- Ptk2 cells stay relatively flat in cell culture, making it easy to see their large chromosomes
- these characteristics made this cell line ideal for studying genetics and the cell cycle (+ they grow nicely in culture and are a great cell line to use in BIOL 2020)
- RG1 cells => can be used in our lab
Lab 1 guide
What cell culture equipment was mentioned in Lab 1? (No specific details)
- Biological Safety Cabinet
- CO2 incubator
- Inverted microscopes
- Media
- Culture vessels
What is a biological safety cabinet?
NOT A FUME HOOD
- needed for a CL2 lab
- protects us from infectious materials or toxins AND protects specimens from contamination
How does a biological safety cabinet work?
- creates an air curtain across the front opening to prevent aerosols from escaping out the front
- prevents unfiltered air from entering the the working area
- inside: air moves in a constant, streamlined speed and direction => creates a laminar flow that contains airborne infectious agents
- air from the cabinet is exhausted through a HEPA filter
- also maintains a sterile work environment by filtering incoming air through the HEPA filter before it blows across the working surface
Lab 1 guide
What is a CO2 incubator used for?
- required for short-term storage of growing cells
- provides a clean, humidified environment with a constant temp
- supplied with 5% CO2, which maintains the pH at physiological level
Lab 1 guide
How are cells stored long-term?
cells are cryopreserved in liquid nitrogen
- cryoprotectants, such as DMSO, are added to the media used for freezing cells to reduce intracellular ice formation and prevent cell death during freezing
Lab 1 guide
Inverted microscopes
have the lens on the bottom of the microscope and light source above the specimen
- needed as cells are usually growing on the bottom of the flask and there is often condensation on the top
- also, flasks and dishes are bulky and the large stage provides an adequate space to view the vessel
Lab 1 guide
Media (for cell growth)
used to provide nutrients the cell requires
- typically contains about 5-10% fetal bovine serum (FBS) => provides nutrients, hormones and trace elements necessary for cell growth and proliferation
- antibiotics often added to minimize risk of bacterial growth following contamination
- phenol red is added to monitor pH
What does a change in the colour of the pH indicator mean for cell cutures?
its a sign that there is excess of metabolic by-products and that it si 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
Lab 1 guide
What are mammalian cells grown in?
specialized culture vessels that have been treated to allow adherent cells to attach to the bottom surface
- e.g., flasks with screw tops that can be vented or non-vented, plates with individual wells
Many experiments in cell biology test the effect of a _______ _______ on some sort of measurable ________
single variable; phenotype
What kind of pipette should be used for volumes greater than 1 mL?
a pipet boy
What are the two types of stressors we used?
- Oxidative stress; from hydrogen peroxide
- pH stress (i.e., acetic acid shock); from vinegar
What was the trick with stressing the cells? (i.e., what was the point?)
to induce apoptosis, but not to over stress the cells to cause necrosis
Apoptosis vs necrosis
apoptosis: organized cell death
necrosis: less orderly (splat!)
delicate balance to prevent necrosis
What needed to be done to the negative control group in lab 1?
the same volume of water, to match the volume of stressor, had to be added to ensure everything besides the stress was the same
Total magnification is the product of what?
the ocular and objective lenses
- ocular is 10X
- objective is 10X or 40X
Why was phase contrast microscopy used in lab 1?
small, unstained specimens (like living cells) can be hard to see using brightfield
- phase contrast converts differences in light phase shifts into differences in light intensity
- made possible by aligning a phase plate that sits at the back of each objective with a special filter in the turret
Trypan Blue
a common stain used to determine cell viability
- dead or dying cells with compromised cell membranes allow the blue dye to leak into the intracellular space
- often called an “exclusion test” => living cells with intact cell membranes will exclude the dye and remain a clear/grayish colour
What was the procedure of determining cell viability in lab 1?
- retrieve the 6-well plate after 30 minute incubation in stressor (oxidative stress or acetic shock)
- retrieve two glass microscope slides and label one “T” and one “C”
- put 25 microliters of trypan blue on each slide
- pick up coverslip from well and place it cell side down on drop of trypan blue
- remove any surrounding liquid and put slide on microscope stage
- observe cells at 10X in both brightfield and phase contrast
- use the best setting for you and increase to 40X (if phase contrast, change to Ph2!!)
What were we looking for when we were determining cell viability?
- membrane blebbing: once apoptosis signaling pathways have been initiated, the cell will start to fragment and small vesicles will bud off from the membrane (one of the most defining characteristics of apoptotic cells
- cell shrinkage: as cell fragments, it will become smaller in size (necrotic cells will swell and appear larger than normal)
Hemocytometer and determining cell concentration
hemocytometer: standard way to count cells; has a specialized slide with an etched grid; pipet cells into chamber on top of the grid
- count the cells in the 4 corners (unless cell concentration is super high or low)
- cells per 0.1 microliters = (numer of cells you counted)/ (number of large squares containing counted cells)
- multiply this by the dilution factor ( sample volume plus diluent volume all over the sample volume) to get it back to the actual concentration (without trypan blue)
- finally, multiply by 10^4 to get it in per mL
How are proteins extracted from cell? What are the 2 methods?
proteins are extracted by disrupting the membranes and collecting the resulting liquid supernatant (i.e., cell lysate)
- physical methods of lysis
- chemical methods of lysis
- preparation must stay cold so the proteins do not denature
Physical methods of protein extraction (how they work and what they do)
use external forces to physically break down the membrane to release the cellular components
- liquid homogenization: apparatus shears cells by forcing them through a narrow space. Can be done with a plunger and vessel or in an automated fashion
- sonication: machine uses pulsed, high frequency sound waves to agitate and lyse cells. Sound waves are delivered using an apparatus with a vibrating probe that is immersed into the cell suspension
- manual grinding (mortar + pestle): technique works well to isolate proteins from plant cells. Freeze tissue in liquid nitrogen and then smash to release proteins
What is the challenge with physical methods of protein extraction?
- it’s often difficult to have consistent preparations from one day to the next
- less reproducible
- these methods can generate heat, which will cause proteins to denature
Chemical methods of protein extraction
use detergents and/or hypotonic solutions (low salt) to disrupt the membrane and extract proteins
- RIPA is a commonly used lysis buffer => contains detergent Triton X-100
- Ripa buffer typically includes protease and phosphatase inhibitors
What are the key steps in extracting proteins from adhering cells using RIPA buffer?
- grow cells in flask
- remove media and wash cells with buffer (like PBS) to remove residual media
- add RIPA buffer to flask => ensure all cells come into contact with buffer
- incubate cells in RIPA buffer on ice for 10+ minutes
- transfer lysed cells to a tube on ice and centrifuge to pellet any membranes
- supernatant will contain a soup of the proteins (= cell lysate)
Marion Bradford
developed a quick and easy way to find the total concentration of protein (expressed as microgram per microlitre) in a solution by using a colorimetric reaction
- technique remains one of the most common ways of testing protein concentration
How does the Bradford assay work?
when a dye in the bradford reagent binds to protein, there is a colour change from brown to blue
- intensity of the blue colour is measured using a spectrophotometer set at 595 nm
- assay uses BSA (bovine serum albumin) as a standard to which the unknown samples will be compared
BSA
bovine serum albumin
- used in the Bradford assay technique as a standard to which the unknown samples will be compared
- BSA isolated from cow blood and is often used in research when a generic protein is needed
