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124 Cards in this Set
- Front
- Back
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What are HEK 293 Cells? |
- Transformed human embryonic kidney cells to deregulate cell cycle - distant relatives of Frank Grahams 293rd attempt at this technique |
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What are adherent cells? |
- attach to substrate, tend to grow in irregular shapes with long projections |
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What is a characteristic of a healthy cells in a culture? |
Tend to be more spread out on a substrate |
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How do cells grow in culture? |
- grow in a predictable fashion - short lag time while cells adapt to new environment - divide in logarithmic fashion - cells eventually reach density where a nutrient becomes limiting - growth rate levels off - cells need to be subcultures to a new flask and new media (called passaging/subculturing) |
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What happens when adherent cells (like HEK 293) grow and divide to a high density? |
- forms a solid mono layer over a substrate - called a 100% confluence - some cells stop growing, others grow on top of eachother - pH changes evident by phenol red indicator (acidic- orange) |
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When should cells be subcultures or passaged? How do you know? |
- 80-90% confluent - pH indicator in media drops, need to split cells if confluent or change media if not yet confluent |
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Why must cells be detached from the walls of the culture vessel before subculturing? |
HEK 293 cells are adherent so they must detach from the vessel before they can be passaged! |
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What is the proteolytic enzyme trypsin used for? |
- (produced in the pancreas) - enzymatically digests cell attachment points from vessel walls -used in conjecture with phenol red for pH |
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Why must Trypsin be diluted after cells use? |
- this is done to ensure that trypsin does not continue to digest the cells (if left could kill the membrane) |
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What are LAIs? |
- laboratory acquired infections! (lab /lab related activities) |
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How can LAIs occur? |
via inhalation, ingestion, inoculation, or contamination of skin or mucous membrane |
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How many Risk Groups classifications are there? -Who classifies them? |
- 4! - The Public Health Agency of Canada and the Canadian Food Inspection Agency |
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What is assessed in the Risk Group Classifications? |
- all microorganisms, proteins, and nucleic acids are assessed to determine their risk to the individual/animal and public health , livestock, poultry |
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What is Risk Group 1? |
- Low individual risk and low community risk -ex. YEAST |
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What is Risk Group 2? |
-Moderate individual risk and low community risk! - pathagens are capable of causing serious disease in humans but are unlikely to do so. - Norwalk virus (stomach flu)/ Dal's human cell lines! |
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What is Risk Group 3? |
- High individual risk and low community risk -these pathogens are likely to cause serious disease in humans and animals - ex. SARS and TB |
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What is Risk Group 4? |
- High individual risk and high community risk - Pathogens can produce highly contagious, serious or fatal disease for while there are no treatments or vaccines |
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What are Containment facilities? |
- Refer to the minimum physical containment and operational practices required for handling infectious materials and toxins safely in the labratory |
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How many Containment Facilities are there? |
- There are 4! |
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What are CL1 facilities? |
- "regular" type of teaching lab - no special design feature other than functional working space and cleanable work surfaces -Biological safety cabinets are not required |
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What are CL2 facilities? |
- Common facility in hospitals and universities used for research purposes! - generally RG2 are contained in CL2 facilities - have HEPA filters -Dal's tissue culture rooms are Cl2 |
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What are CL 3 facilities? |
- require additional and secondary barriers to minimize release of infectious organisms into the environment? - Includes sealed windows that use a biological safety cabinet for all work with strict controlled access! |
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What are CL4 facilities? |
- maximum level of biosafety and biosecurity -complete seal of the facility perimetere, including electrical and plumbing - Lab workers must wear full coverage protective- pressure suit with own breathing supply - Chemical showe before removing protective clothing - Only 2 sites in Canada! |
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What is the most critical piece of equipment needed for working in a CL2 lab? |
- The biological Safety Cabinet, also called the HOOD! |
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What is the purpose of the BSC? |
- protects you from infectious material and toxins - ALSO protects specimens from contamination |
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How does a Biological safety cabinet work? |
- creates an air current across the front opening stops aerosols from escaping - HEPA( high efficiency particulate air) filter is used to filter the air before it is comes into the Hood -UV light |
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What is the purpose of UV light in the BSC? |
- used to sterilize the work area before use |
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What should the hood be wiped down with to sterilize before beginning work? |
- 70% ethanol |
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What conditions are mammalian cells grown in? |
- temperature 37* C - humid environment - 5% CO2 to maintain pH at physiological level |
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How are mammalian cells viewed? |
- Use of an inverted microscope that has the lens at the bottom and light source above - cells stick to the bottom of the flask! |
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How are HEK 293 cells stored longterm? |
-use of Cryopreservation using liquid nitrogen with media containing dimethylsulfozide (DMSO) to help slow the rate that cells freeze to minimize damage! |
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What is included in the media? |
- 10% fetal calf serum (or bovine/horse) - Penicillin and Streptomysin is added to minimize the risk of contamination -Phenol red used as pH indicator (orange if acidic, purple if basic) |
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What culture vessels were used in the experiment? |
- Culture flasks T-25, vented to allow for gas exchange |
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What is a Hemocytometer? |
- specialized slide with a grid on it that allows for cells be counted in "blocks" - is helpful when determining density of cells/ mL |
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Before an experiment what needs to be done to prepare the Biological Safety Cabinet? |
- 70% ethynol used to clean work area -new pipettes and empty T-25 placed in hood - UV turned on for 10-15 min |
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What is removed from the 37*C incubator before use in the BSC? |
- media (includes HEK cells), trypsin and PBS are taken out and bottles are sterilized! |
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At what confluence should cells be split/passaged? |
80-90% |
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What is PBS used for? |
- helps to get rid of calcium build up on cells |
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How can you tell that HEK cells have released from the bottom of a T25 flask? |
- cells will be visible floating around in the liquid! |
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What is Trypan Blue used for? |
- used to determine cell viability by staining cells! - dead or dying cells have a compromised cell membrane which allows the dye to penetrate the cell body - called the EXCLUSION test (as living cells will remain clear/grey) |
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Phase Contrast (Ph1) usually makes what kind of cells easier to find? |
- alive (unstained) cells are easiest seen on this setting! |
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Bright Field (O) is usually best to view what kind of cells? |
- best used to find stained dead or dying cells! |
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How can cell concentrations be determined??? |
Cells per ml= (#cells counted/ # of squares examinied )* dilution factor* 10^4 |
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What is apoptosis? |
- A type of programmed cell death where a cell self destructs in response to cell signallying! - process is highly controlled, orderly proceess that happens in healthy, normally functioning cells! |
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What is the requirement of an immortalized cell? |
- any cell line that has the ability to divide indefinitely! - some can be deliberately manipulated (HEK293) and naturally derived cancer cells (HeLa) |
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How is Kohler Illumination attained? |
-turnet set to O - focus at 10x, -close field iris completely - move substage condenser under the stage -center the ring -center the field iris |
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What was the purpose of the concentrated hydrogen peroxide used in the experiment? |
- used to cause oxidative stress in the cells! |
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How long were cells incubated in control or hydrogen peroxide conditions? |
-30 minutes! |
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What is the purpose of lysing the cells? |
-used to burst membranes and extract proteins! |
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What was the purpose of the Bradford Assay? |
- used to prepare samples of equal protein concentrations |
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How can apoptotic cells be recognized after exposure to hydrogen peroxide? |
- morphological changes like blebbing will be observed! -will see a cell size/shape change - will be stained with trypan blue if dead! |
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What does 0.75 ug/ul mean? |
- 0.75 ug in 1 ul of liquid! |
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How does a bradford assay work? |
- using colorimetric reaction to determine the protein concentration - dye in bradford reagent binds to proteins in acidic medium, colour changes from brown to blue! - intensity of the blue is measured using a spectrophotometer at 595nm |
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Why are experimental samples tested against a set of known protein concentration samples for a bradford assay? |
- used an measured to give a basis for comparrison! |
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What is Bovine Serum Albumin (BSA) used for in the lab? |
- Used as a standard for protein concentrations to which unknowns can be compared to using a line of best fit! - linear between 0.20-1.0 ug/ul |
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Why do we need to dilute the cell lysate to the same concentration? |
- needed for electrophoresis to ensure that equal amounts of cells are loaded into wells for a fair comparison! |
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How long does the bradford reagent test take for the reaction to take place? |
-about 10 minutes! |
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What information is collected from the spectrophotometer for the experiment? |
the absorbance values from the bradford assay is crucial as this is later used to determine the protein concentrations in the sample cell lysates1 |
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What is an important shared characteristic between control and treatment cell lysates needed in order to compare protein expression? |
- need to run the same TOTAL number of micrograms in each lane! Samples need to be diluted to the same concentration!!! |
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When making dilutions, what formula can be used? |
C1V1=C2V2 |
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What is the use of gel electrophoresis? |
-used to separate proteins by molecular weight! |
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What is the purpose of staining the gel with Coomassie Blue? |
- stains the proteins of the entire protein set so they can be examined |
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What is the purpose of transferring the gel onto nitrocellulose membrane? |
- Western blot can be used to detect specific proteins! |
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How does electrophoresis work? |
-commonly used to separate charged molecules (like DNA and proteins) using an electric field |
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How does the gel allow for protein/DNA seperation? |
- uses a molecular sieve that seperates molecules based on molecular weight! |
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What kind of gel are proteins generally run through? |
- polyacrylamide |
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What kind of gel is DNA normally run through? |
-Agarose |
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Can proteins be seen running through the gel? |
- no they cannot be seen, they are later stained with Coomassie Blue! |
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Is Uv light used to view proteins in a gel? |
- no, the use of UV is only used in DNA gels! |
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What is the purpose of sodium dodecyl sulfate (SDS)? |
- disrupts the secondary structure of the molecules so that proteins become dematured and rod shaped! -movement through the gel does not depend on protein shape! -also coats with negative charges so that natural charge by R groups is masked! Allows for mobility to be strictly dependent on molecular weight! |
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What size proteins will migrate more rapidly and subsequently further in the gel? |
- smaller polypeptides have an easier way through the pores, and therefore migrate more rapidly than large pores! |
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Besides the use of SDS, what is done to ensure that the proteins have denatured? |
- proteins can be boiled for several minutes - treated with reducing agents like Mercaptoethanol or DTT to break disulfide bonds!. |
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How can gel electrophoresis be used to determine the molecular weights of proteins? |
- known molecular weights can be used, known as "standards" - The distance migrated can be plotted as a function of log molecular weight to create a standard curve! |
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How can sample protein molecular weights be determined? |
- using distance migrated in sample proteins compared to graphed known protein distances allows for an estimation of their molecular weight |
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What molecular weight marker was used in the experiment? |
- Pink plus pre-stained protein ladder was used - uses 11 proteins from 175- 10.5 kDa! |
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What is added to gel electrophoresis to determine where the proteins are? |
- tracking dye in sample buffer is used to track how far the sample has run through the gel! |
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What is added to the cell lysate proteins before they are run in gel electrophoresis? |
- sample buffer- (contains SDS, glycerol, meracatothanol, and tracking dye) |
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What percent acrylamide concentration are the gels that were used? |
- 12% and only 1.0 mm thick! |
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What is the purpose of MOPS SDS running buffer? |
??????? |
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What is the destaining solution used for? |
-Removes Coomassie blue from the acrylamide gel where there are not proteins! |
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How are proteins transferred from Acrylamide gel to nitrocellulose membrane? |
- using a Bio-Rad Trans-Blot Turbo machine, uses a current to pass through the gel and move the negatively charged proteins out of the gel onto the nitrocellulose! |
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What is used to prevent the polyacrylamide gel from drying out? |
- Running buffer |
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In which direction do the proteins move from the gel towards the nitrocellulose? |
- Gel is placed UNDER the pre-soaked nitrocellulose, and proteins therefore move up! |
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What should the nitrocellulose membrane be stored in? |
TBS-Tween! |
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Why is it important to measure the distances that the standard ladder migrated through the gel? |
- used to create a standard curve that can be used to determine the molecular weights of the tested HEK cell proteins |
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What is another term for western blotting? |
- also called immunoblotting |
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How does western blotting/ immunoblotting work? |
- uses antibodies to probe for one specific protein on the nitrocellulose membrane! |
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What are antibodies? |
- important proteins produced by an animals immune system! Can be used specifically to target specific proteins (called antigen) |
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How are antibodies made? |
- raised and collected from an animal host, often mouse, rabbit, or goat! |
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Antibodies used belong to the IgG class, what shape structure do they have? |
- Y shaped strucutre! |
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The base of the Y represents (on antibodies)? |
the constant domain! - same in all antibodies from one particular animal! |
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The branches of the Y represent (on antibodies)? |
- variable region! - compliment the shape of the antigen and allows for binding to occur! |
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How are antibodies generated? (Polyclonal antibodies) |
- traditionally used by injecting an animal host with purified antigen/protein of interest! - animals immune systems will produce a variety of antibodies in response - the antibodies can then be collected from the animals blood and be sorted by specific epitome of the protein of interest |
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How are monoclonal antibodies created? |
-all identical to each other and are specific for the same epitope on an antigen! -produced by injecting a mouse with antigen, and removing some of the antibody producing cells! -one of the cells in the culture is then cloned!!! -the antibody is isolated from the cultured cells and produce a single colony of cells. -These antibodies are very specific and less likely to bind to other random proteins non-specifically! |
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What are secondary antibodies? |
- binds to the primary antibody - the organism that produces primary cannot also produce secondary! |
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How are secondary anti bodies named? |
- host organism: chicken - primary from rat - CALLED chicken -anti rat! |
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Secondary antibodies have what added benefit from their use? |
- often have a chemically linked reporter molecule - can be an enzyme, dye, or fluorochrome |
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What is the enzyme attached to the secondary antibody used in the experiment to catalyst a light-producing reaction? |
- horseradish peroxidase (HRP) |
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When there is a reporter molecule attached to a secondary antibody how is it named? |
Ex: Chicken anti-rat, conjugated to HRP (remember chicken is host, rat is where the antigen was and the reporter molecule is added at the end!) |
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What is chemiluminescence? |
- emission of light due to a chemical process |
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Why is chemiluminescence used? |
- used to determine the location of our target protein on the nitrocellulose! |
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What reaction does the enzyme horseradish peroxidase (HRP) catalyze? |
- luminol and oxidizing reagent!, oxidation of luminol occurs and light is released! |
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What wavelength of light is released from the oxidation of luminol |
- 428 nm, which can be captured on blue autoradiography film! (but is not intense enough to be seen by the naked eye) |
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What is Ponceau stain used for? |
- used to temporarily stain the proteins on the nitrocellulose so the location of the protein lanes can be identified! |
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What is the purpose of the milk solution (5% milk power in TBS)? |
- coats nitrocellulose in casein (milk protein) and helps to prevent non-specific binding of the primary antibodies to the nitrocellulose where there are not HEK proteins |
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What is TBS-Tween used for? |
used as a buffer to rinse the membrane! |
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What is applied first to the membrane? Primary or secondary? |
-Primary is applied first! |
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If Primary was raised in rabbit, what is the secondary anti-body needed? |
- (animal host)- anti rabbit antibody! |
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What is the use of the standard curve created from the coomassie stained gel? |
- used to calculate the molecular weight of the various bands in your samples! |
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What is the purpose of Immunofluorescence? |
- another method that can be used to look at protein expression and to visualize cell processes! - used to determine if a protein is expressed in a particular cell and where it is located |
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What is another word for immunofluorescence is? |
- immunohistochemistry |
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Are primary and secondary antibodies used in immunofluorescence? |
- Yes! the secondary antibodies in the lab are tagged with fluorochromes (instead of HRP for luminol) and emit light in the visable spectrum! |
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Fluorochromes/fluorophores can emit different wavelengths how? |
- absorb light for a short time and then re-emit it at a slightly different wavelength! |
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What is the role of the microscope lens in immunofluorescence? |
- different lenses act like a filter that allows only light of the desired emitted wavelength to pass! - This light forms a coloured image |
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What is the function of FITC? |
- it is a fluorochrome, conjugated to the secondary antibody and used to label tubulin in the microtubules! |
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What is Hoechsts? |
- used to stain the nucleaus, but does not interact with antibodies at all, rather interacts with DNA! |
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How does labelling of microtubules work in immunofluorescence? |
- primary antibody recognizes tubulin! Secondary antibody attaches to the primary conj. with FITC, emitting green light!!! |
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Hoes does labelling of the nuclei work in immunofluorescence? |
- fibroblast nuclei will be labeled blue using fluorochrome HOESCHST! - Binds directly to the DNA of the cell, so does not require antibodies! |
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Immunofluorescence can be used to label any part of the cell as long as you have appropriate? |
- Antibodies! - If labelling the mitochondria? - use a primary antibody that is specific to ATP synthase (mouse anti- ATP synthase) |
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What colour is Fluorochrome Alexa Fluor 555? |
- seen as red! |
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What is the purpose of methanol? |
-fixes the cells so that the cytoskeleton stays intact and helps the cells stick to the coverslip! -also permeabilizes the cell membrane! |
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What is the purpose of humidity chambers used in lab 5? |
- provide a moist environment uring antibody incubation so the cells do not dry out! |
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The negative control had no primary antibody in lab 5, if there HAD been green glowing in the microcopy? |
- contaminated with primary - OR poorly make secondary antibody that is binding not to the primary but sometjing directly in the cell |
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What does gycerol do? |
- needed to cause sample protein lysates to sink to the bottom of the well in the gel! |
