Chapter 1: Abbreviations
American Type Culture Collection Rosewood Park Memorial Institute Hank's Balanced Salt Solution Fetal Bovine Serum Phosphate Buffered Saline Dulbecco's Modification of Eagle's Medium
What does D10 constitute of?
89% DMEM 10% FBS 1% Glutamine
List the advantages of
1) Control of environment 2) Better use of reagents 3) Other issues (Ethical, moral and legal)
List the disadvatages of
1) Expertise needed 2) Quantity and cost (of equipment)
What does the culture environment affect?
1) The nature of phase on/in which the cell grows (Adherent or suspension) 2) Medium 3) Constitution of gas phase 4) Incubation temperature
What is the process of cell adhesion?
1) Cells secrete extracellular matrix (ECM) 2) Matrix adheres to charged substrate 3) Cell then binds to the matrix through specific receptors
Name the three adhesion molecules, and list their functions/properties.
Cadherins Interactions between homologous cells Calcium dependent
Integrins Interactions between cell and substrate Binds to matrix molecules via RGD motif
Transmembrane proteoglycans Interactions between cell and substrate DOES NOT bind to matrix molecules via RGD motif
How does cells metabolise energy?
Glycolysis and Citric Acid Cycle
What are the properties of media?
pH CO2 and Bicarbonate Buffering Oxygen Temperature
What is the make up of complete media?
Amino Acids Vitamins Salts Glucose Antibiotics Serum
What is the function of amino acids, and what are the amino acids needed in complete media?
The concentration of AA influences the cell survival and growth rate of the cells
Essential AA: Isoleucine, leucine, lysine, Methionine, Phenylalanine, Threonine, Tryptophan, Valine Cysteine Tyrosine Glutamine
What are the types of vitamins in complete media?
Fat soluble (A,D,K,E) and water soluble (Choline, folic acid)
Name the salts and their functions in complete media.
Ca2+ --> Cell adherence (cadherins) Na+,K+,Cl- --> Regulates membrane potential SO42-, PO43-, HCO3- --> Regulates intracellular charge
What is glucose for in complete media?
What is the function of antibiotics in complete media? List the advantage(s) and disadvatage(s).
Advantages Reduces contamination
Disadvatages - Antibiotic resistant strains of organisms may arise - Hides low level contaminants that may appear when antibiotics is removed - Encourages poor aseptic techniques - Antimetabolic effects
What does serum contain?
Growth factors (eg. Platelet derived growth factor PDGF, Fibroblast growth factors FGF, Epidermal growth factor EGF) Adhesion factors (eg. Fibronectin) Proteins (Major componet in serum. Carriers for other molecules) Hormones (eg. Insulin and Hydrocortisone- cell attachment and proliferation) Lipids (eg. Oleic acid) BOUND TO PROTEINS Minerals (eg. Iron, copper, zinc) BOUND TO PROTEINS
What process is used for making media? What is the material used?
Filtration using a 0.22um filter.
What is the process of producing antiserum?
1) Inject animal with substance of interest (usually mixed with adjuvant) 2) After 1 month, a booster is given 3) Two weeks later, blood is drawn and serum is harvested
What is the difference between the first antigenic challenge and the second antigenic challange?
The second antigenic challenge is:
More immediate in onset Of a higher titer Higly specific for that antigen Persists longer
What is polyclonal antiserum?
Polyclonal antiserum contains antibodies that bind to an antigen, but at different binding sites or affinities. This is because they have differenct hypervariable regions (where they bind to the antigen).
Where do antibodies come from?
B cells that are primed to proliferate turn to plasma cells which make antibodies. Cytokines drive differentiation and proliferation of B cells to become plasma cells.
What are monoclonal antibodies?
Monoclonal antibodies come from the same B cell. They bind to the same antigen at the same site and at the same antigen binding site. (They are all the same!)
What are hybridomas?
A hybridoma is a cell line that produces monoclonal antibodies and is immortal. (Basically Spleen cells+Myeloma Cells)
What does PEG and HAT stand for and what do they do?
PEG - Polyethylene glycol fuses myeloma and spleen cells HAT - Hypoxanthine aminopterin thymidine Blocks a pathway in myeloma cells so they die. Myeloma cells don't have the enzyme HGPRT while spleen cell does, but spleen cell dies within 2 days.
What are 2 hosts that you can use?
Balb/c mice and rat.
How to choose myeloma cell line?
The cells must not synthesise immunoglobulin, must die in selective HAT media, and must be immortal.
What are some kinds of immunogens?
Purified proteins, glycoproteins, carbohydrates, recombinant proteins, whole cells.
Why do you use an adjuvant to prepare the immunogens, and what is the adjuvant to use?
Adjuvant is use to augment the immune response. A common adjuvant to use is Freund's Complete Adjuvant (FCA).
What is added to the medium to eliminate revertant myeloma cells containing normal levels or HGPRT?
What is the ratio and concentration of myeloma cells to spleen cells?
Myeloma cells 1x10^7 and Spleen cells 5x10^7
How do you obtain wells with hybridomas that make the desired antibody?
What is HAMA?
Human Anti-mouse Antibodies
What are uses of monoclonal antibodies?
Research (analysing cell surface markers and Intra cellular markers) and Clinical Applications (Diagnostic and Therapeutic)
2 types of hybrid monoclonal antibodies are:
Chimeric antibodies (human constant, mouse variable) and humanised antibodies (Human antibody with mouse hypervariable regions)
What is the difference between bispecific antibodies and bifunctional antibodies?
Bispecific antibodies are antibodies that are attached to antibodies
Bifunctional antibodies are antibodies that are attached to other molecules (eg. toxin)
Why carry out cryopreservation?
Avoid genomic and prototypical changes Avoid cross contamination by other cell lines Incubator failure Maintaining cell lines which are not for immediate use For distribution
What happens if freeze rate is too slow or too fast?
Slow - Water move out of cell Fast - Water will crystalise and damage cells
What is the procedure for freezing cells?
Step 1: Harvest cells Step 2: Place cells in freezing media (growth media + 10% FBS + 10% DMSO or glycerol) Step 3: Place in vials Step 4: In -70C Step 5: In -196C
Name some possible sources of contamination
Operator technique Environment Use and maintenance of laminar-flow hood Humid Incubators Cold stores Sterile materials Imported cell lines and biopsies
How to monitor for contamination?
Eye Microscope Gramstaining on culture plates
How do you confirm mycoplasma contamination?
DNA fluorescent staining (Hoechst) PCR ELISA Immunostaining
What are the visible signs of contamination?
Sudden change in pH Cloudiness in the media Granules, ovoid particles and filamentous mycelia seen under microscope
What are ways of detection for viral contamination?
What are the steps for eradication of bacteria, fungus and yeast?
Wash cultures several times in high concentration antibiotics Grow the cultures for 3 subcultures in media with antibiotics Further cultures for a further 3 subcultures without antibiotics Check to ensure contamination is eliminated
If its for mycoplasma, use Mycoplasma Removal Agent (MRA)
What are two ways to count cells?
Use a Hemocytometer or a Electronic Counter
What are the formulas to calculate: percentage viability, viable cell concentration and total number of cells?
(Count 4 big squares and take the average)
%viability = live cells/live and dead cells x100%
Viable cell concentration (cells/mL) = average number of life cells x dilution factor x 10^4
Total number of cells = viable concentration of cells x volume
What is a primary cell culture, and what are the sources used to obtain them?
Primary cell culture are cells that are first obtained from the source. Sources are mouse embryos, human biopsies, and chicken embryos.
Name 3 techniques used to obtain cells for primary cell culture.
Dissection (Migration) (Chop tissues to fine pieces and then seed in flask) Mechanical Force (Sieves, syringe+needle or vigorous pipetting) Enzymatic Disaggregation (Trypsin, Pronase, Collagenase and Dispase)
What does the actual number of doublings depend on?
Species differences Lineage differences Culture conditions
How do you make a finite cell line continuous?
Transfect with Telomerase, oncogenes, interactions with SV40 genome/whole virus
What is passage number and generation number?
Passage number - The number of times a culture has been subcultured
Generation (Doubling) number - Number of doublings a culture has undergone