TI Flashcards
What is Primary Cell Culture?
A technique where cells are isolated from the body tissues and grown in culture, trying to create in vivo conditions.
What are examples of non-haemopoeitic primary cultures?
- Liver
- Muscle
- Fibroblasts
- Skin
- Nerves
- Endothelial Cells
What are examples of haemopoietic primary cultures?
- Stem, progenitor cells
- Monocyte, macrophages
- T and B cells
- Dendritic cells
- Osteoblasts
- Erythrocytes
- Megakaryocytes, platelets
- Neutrophils, Eosinophils, Basophils, Mast Cells
What are the 6 features of primary cell cultures?
- cells derived directly from tissues
- interpatient variability
- finite lifespan
- cells divide and/or differentiate
- cells carry out normal function
- cell lines
What happens in the disaggregation of cells?
- cells are allowed to migrate out of an explant
- mechanical dissociation
- enzymatic dissociation
What type of cell doesn’t need to be disaggregated in primary cell culture and why?
Haemopoietic cells because they are already disaggregated
What are the sources of stem cells?
- Bone marrow
- Umbilical cord blood
- Mobilised peripheral blood
What is the path from stem cells to blood cells?
Stem cells... Early progenitors... Late progenitors... Immature precursors... Red cells/neutrophils/platelets!
What do the stem cells that sit out of the cycle do?
either divide to produce identical clones of themselves or differentiate
At what point do stem cells become visually distinguishable?
immature precursors
What does CFU stand for?
Colony forming unit. (the letters at the end show which lineages they are limited to)
What are the constituents of bone marrow?
- Areas of tightly packed cells - progenitors and early stem cells
- Blood vessels
- Fat
What are the defining features of stem cells?
- pluripotent
- self-renew
- rare
- responsible for engraftment
What are the defining features of progenitor cells?
- undifferentiated
- undistinguishable by morphology
- committed to one or more lineages
- detected in colony forming assays
What are the defining features of precursor cells?
- immature but recognisable
- starting to differentiate
- few final divisions before they become mature cells and move into the peripheral blood
What do haematopoeitic growth factors do?
They are polypeptide growth factors, also known as cytokines.
They bind to the cell surface transmembrane receptor and stimulate growth and survival of progenitors.
About the structure of stromal cells…
they have an extracellular matrix and adhesion molecules on their surface. They provide cytokines.
Why do stem cells need to be processed?
stem cells are few in number and so we want to enrich and purify them
What are processing methods of stem cells?
- Erythrocyte lysis - ENRICHMENT
- Density gradient centrifugaton - ENRICHMENT
- Adherence depletion - ENRICHMENT
- Antibody depletion - PURIFIED STEM CELLS
- Antibody selection - PURIFIED STEM CELLS
Why are progenitors called CFUs?
Because they grow to form colonies of mature cells, containing up to 1000s of cells.
What do you need for a colony assay?
- semi-solid medium (eg agar)
- growth factors
What happens in colony assays?
Larger progenitors are forced to mature and divide into colonies using growth factors when put into culture on a semi-solid medium. We can quantitate the number of CFUs we had originally and can use a microscope to identify the different CFUs.
What is LTBMC?
long-term bone marrow culture
What are the steps of LTBMC?
- bone marrow
- ficoll gradient
- liquid culture
- CFU
What happens in LTBMC?
We use flasks in which the stromal cells are at the bottom. The stem cells bury into the stromal cells, which stimulates them to divide. The stem cells emerge from the stromal cells as committed cells (stem cells remain in the stromal cells) and move into the supernatant. There is a direct relationship between early stem cells and later progenitors.
What are the applications of primary cell culture techniques?
- Research - basic haematopoeisis and carcinogenesis
- Testing toxicity of chemotherapeutic agents and carcinogens
- Generate cells for stem cell transplantation/manipulation
How are cells isolated from the blood?
- Density centrifugation
- Fluorescence activated cell sorting (FACS)
- Immune-precipitation
How are cells isolated from solid tissue?
Mechanical and enzymatic disruption using collagenase, dispose and trypsin
What are the pros of primary culture?
Unmodified
What are the cons of primary culture?
- aberrant expression of some genes
- variable contamination
- poor growth characteristics (50-100 divisions)
- inter-patient variability
- phenotype instability
- molecular manipulation is difficult
What is the idea model for cell culture?
- Good growth characteristics
- Phenotype stability
- Defined population
- Molecular manipulation readily achieved
What are the two ways in which cell lines can be derived from primary culture?
- Spontaneous - from tumours or prolonged culture, multiple ill-defined mutations, transformed phenotype
- Genetic manipulation
What do we target to generate cell lines?
Processes that regulate growth and ageing…
- p53
- retinoblastoma gene (Rb)
- telomerase
What is the role of p53 in a normal ageing cell?
- telomere binding protein protects the telomere ends (TTAGGG)
- when the telomere gets too short, the binding protein is lost
- p53 binds to the unprotected chromosomes
- activated p53 triggers growth arrest or cell death
What is the t-antigen?
A viral protein which interacts with normal p53 and Rb. This can cause increased growth without loss of function.
What is HPV/what does it do?
A viral protein - E6 targets p53 for degradation, E7 binds to Rb
What viral genes are commonly used to generate cell lines?
- SV40; T and t antigen
- HPV; E6/E7
…p53 and Rb bind to different viral proteins
What is the role of telomerase?
Telomerase prevents the erosion of telomeres. Some cells need telomerase and silencing of Rb for ‘immortalisation’. E6/E7 and telomerase are believed to maintain a differentiated phenotype.
What are the steps in the derivation of cell lines?
Plasmid…Gene for selection…Growth promoting gene
Transfection
Selection pressure added
Colonies selected
When generating cell lines, what hurdles have to be overcome?
- getting the DNA into the cells
- getting the cells to stably incorporate the DNA once inside
What are the 5 methods of transfection?
CaPO4 co-precipitation Lipofection Electroporation Viral transfection Nucleofection
What is lipofection?
- positively charged lipoplexes interact with the negatively charged cell membrane
- taken up by endocytosis
- release from endosome
- transport to the nucleus
- entry to the nucleus is inefficient and may need mitosis
What is electroporation?
An electrical field is applied to the cells in order to increase permeability of the cell membrane, allowing chemicals, drugs or DNA to be introduced to the cell.
The pores reseal when they have been created by the electrical current, and the rate of this resealing is dependent on temperature.
What is viral transfection?
- Exploits the normal mechanism of viral infection
- Usually has high transfection efficiency
- There are three gene-targeting vectors commonly used based on three types of viruses; retrovirus, adenovirus, and adeno-associated virus.
What is nucleofection?
- combination of electroporation and lipofection
- increased efficiency particularly of non-dividing cells
- technology is protected under patent
- different solution and protocols are used for each cell type
What are the disadvantages of cell lines?
- RAPIDLY DIVIDING CELLS OFTEN LOSE DIFFERENTIATED FUNCTION
- therefore in making cell lines, growth and function are at odds
- the ideal solution would be a cell line that divides when you want more and stops when a study of function is requited
What are the means to overcome loss of function of cells in cell lines?
- conditional mutant eg T-antigen of SV40
- changing culture conditions such as the ECM, co-culture or 3D culture
What is 3D culture?
An artificially created environment in which biological cells are permitted to grow and interact with their surroundings in all three dimensions
What are the advantages of 3D cultures?
- genes expressed as in vivo
- cell-cell communication is re-established
- cells are orientated in the same was as in tissues
What are the three ways of Light Microscopy?
- histology
- phase contrast
- time lapse
What is Stoke’s Shift?
Due to energy loss, the emitted light is shifted to longer wavelength relative to the excitation light. It is the difference between the two peaks on an excitation-emission graph.
What is photobleaching?
Bleaching of fluorochromes - due to high intensity illumination the flurophores might permanently lose their ability to emit light
How can you avoid photobleaching?
- Work with reduced excitation light intensities or use grey filters
- Use shorter exposure times/higher gain settings and longer intervals during time lapse studies
- Use anti-bleach in your mounting media
What are fluorescent proteins?
These proteins are naturally found in light-producing cells of cnidarians. Fluorescent proteins can be fused with other proteins and introduced in cells via transfection. This allows live study of fluorescent tags in living cells/organisms.
What is the benefit of confocal microscopy over wide field microscopy?
Higher z-resolution and reduced out-of-focus-blue make confocal images crisper and clearer
What is the negative of confocal microscopy compared to wide field microscopy?
Only a small volume can be visualised by confocal microscopes at once. Bigger volume need time consuming sampling and image reassembling.
What are the characteristics of stem cells?
- can self-renew
- can differentiate into a range go more specialised cells
What is meant by potency of stem cells?
Their ability to differentiate
What is multi potency?
can only form one type of cell - i.e. HSCs can only form blood cells, but various types of blood cell
What is totipotency?
Can produce all of the differentiated cells in an organism. Can give rise to the embryo and the tissues that support its development, such as the placenta. These supporting tissues are known as the trophoblast.
What is pluripotency?
Can give rise to all the cell types that make up the body. This is similar to totipotency but don’t make the supporting tissue, only the embryo.
What do multipotent stem cells do for the body?
(aka Adult stem cells) function as a repair system for the body, replenishing old or damaged tissues
What is asymmetric stem cell division?
produces one stem cell and one differentiated cell. QUIESCENCE
What is symmetric cell division?
Produces 2 stem cells. ACTIVATION - leads to expansion of the stem cell pool and occurs due to injury/disease
What is the stem cell niche?
This is the area in which stem cells reside. It is the microenvironment where you find the stem cells, and it interacts with the stem cells to determine their fate. It also regulates stem cell activity.
The niche helps to maintain the balance between symmetric and asymmetric stem cell division.
What is the Extracellular Matrix in terms of adult stem cells?
- key component of the stem cell niche
- two way interaction; stem cells also remodel the ECM
- critical regulator of stem cell function
- mechanical features can determine cell fate
…brain stiffness induces neuron formation…
…muscle stiffness induces muscle formation…
…bone stiffness induces osteoblast formation…
How is the lifespan of a stem cell extended?
- can undergo more cell divisions than somatic cells
- length of telomeres determines how many divisions can occur
- have high expression of telomerase which rebuilds the telomere
What is the therapeutic potential of adult stem cells?
- have clinical applications
- can be autologous - won’t be rejected
- fewer safety concerns - no need to immunosuppressants, not linked to tumour genesis
- easier to get sufficient numbers
Where are Mesenchymal Stem Cells (MSCs) found?
Primarily found in bone marrow but also in... placenta umbilical cord blood adipose tissue adult muscle corneal stroma dental pulp
Why do MSCs have therapeutic potential?
- homing capacity - tend to home to damaged tissue sites
- high differentiation potential
- production of trophic factors
- immunomodulation
What is osteogenesis imperfecta?
Also known as ‘brittle bone disease’, this is a genetic disorder in which osteoblasts produce defective type I collagenase. Symptoms include osteopenia, multiple fractures, severe bony deformities and shortened stature.
What are limbal stem cells?
Corneal renew and repair are mediated by stem cells of the limbus.
What is transdifferentiation?
The process by which an adult cell can change from its initial differentiated state to another. This is largely an in vitro phenomenon.
What are cancer stem cells responsible for?
The growth of tumours is fuelled by limited numbers of dedicated stem cells that are capable of self-renewal. Many studies have suggested that CSCs are resistant to cancer therapy which could explain relapse as the CSCs can relocate and form a tumour in the new location. Therefore, if we target therapeutics specifically to CSCs we may be able to cure the cancer permanently.
What can we use to target cancer stem cells?
- surface markers
- signal cascades
- ABC cassette
- the microenvironment
What does MRD stand for?
Meaningless Random Differences
What does DWC stand for?
difference is worth considering
What does SUD stand for?
statistically unreliable differences
What does FCD stand for?
fairly convincing differences
What is the advantage to the visual approach to data analysis?
Immediate visual summary of effect size and confidence
What are the disadvantages of the visual approach to data analysis?
- does not give a precise measure of confidence
- comparing one confidence interval to another cannot be translated into a numerical unit
What are the advantages to using p-values?
gives a single number that summarises whether your data looks random
What are the disadvantages of using p-values?
- very little information
- doesn’t tell you effect size
- doesn’t tell you confidence levels
What is a type 1 data error?
When we get p
What is a type 2 data error?
When we get p > 0.05 even though there is a real effect. This often occurs when you haven’t repeated the experiment enough, or your sample size is too small.
What should you do if the data differs substantially from the normal distribution?
- mathematical transformation e.g. take a log of the data
- non-parametric test
- Mann Whitney U test instead of T-test
- Wilcoxon signed ranks test instead of paired T-test
What should you do when comparing more than two groups?
- not valid to do a series of t-tests for each pair
- multiple testing increases the frequency of the type 1 errors
- an alternative is to use 1-way analysis of variance (ANOVA)
What does an ANOVA test do?
Looks at data from all groups and asks how likely or unlikely it is that the data as a whole is random. ANOVA compares the variance within each group to the variance between groups.
The variance within each group gives you a measure of random variation.
In ANOVA testing, if the variance between groups is small compared to within groups what does this suggest?
This suggests that the different between groups could be explained by random variation.
In ANOVA testing, if the variance between groups is large compared to within groups what does this suggest?
This suggests that it is unlikely this can be explained by random variation.
What does it mean if the p-value for ANOVA is less than 0.05
- unlikely the differences between groups is random
- valid to ask which differences are responsible for this
- can do individual tests between each pair of the groups
- t-tests or other ‘post hoc’ tests
What is pluripotency?
the ability of a cell to give rise to every tissue in the body
What are the two types of pluripotent stem cell?
embryonic stem cells (ES) and induced pluripotent stem cells (iPS)
What are the stages of derivation of an ES cell?
- cleavage stage embryo
- cultured blastocyst
- isolated inner cell mass (grown on layer of irradiated mouse fibroblast feeder cells)
- cells dissociated and replated to avoid overgrowth (and new feeder cells)
- established ES cell cultures
what is the hinderance of ES cell lines?
only 10% of embryos produce these stem cells
The adult body is formed from a single cell (the oocyte) by…
- migration
- cell division
- differentiation into different types (tissues)
- apoptosis
what drives differentiation of ES cells?
proteins secreted by cells called growth factors
What are the stages of pancreatic development?
- formation of the GUT TUBE
- formation of the dorsal and ventral BUDS
- BRANCHING
- endocrine progenitors MIGRATE in mesenchyme, where they differentiate and form ISLETS
What do the pancreatic islets contain?
the insulin-secreting beta cells
What are current projects of tissue engineering?
- biodegradable polymers for nerve regeneration
- hydrogels for musculoskeletal repair
- electrospun fibres to allow generation of artificial tendons
- windpipe made of glass
What are therapeutic targets of regenerative medicine?
- heart disease
- type I diabetes
- CNS trauma
- neurodegenerative disease
What are the disadvantages of embryonic stem cells?
- ethically contentious
- transplantation would involve immunosuppression
- immune matching involves generating >1000 variable ES cell lines in the UK
- far more difficult to derive than first anticipated
- currently 300+ human ES cell lines world wide and very few clinical trials
What are induced pluripotent stem cells (iPS)?
a type of pluripotent stem cell that can be generated directly from adult cells - formed when you force the expression of 4 genes in fibroblasts (skin cells)
What are the yamanaka factors?
When first investigating iPS cells, scientists found there were 3 genes that caused complete loss of colonies when they weren’t included:
- factor 14 (oct 4)
- factor 15 (sox 2)
- factor 20 (klf 4)
They also noticed that removal of cymc (factor 22) allowed colonies to form but they didn’t look right.
They went on to show that these 4 factors alone could cause the formation of ~160 colonies, whereas the most a 3 factor combination could form was ~50
These 4 factors are known as the YAMANAKA FACTORS and are still used today.
What are methods of pluripotency assessment of iPS cells?
- compare cell morphology
- look for expression of pluripotency associated genes using PCR
- check for expression of markers by fluorescent staining
- look for proteins that should be expressed in the nucleus (oct 4, Nanog and sox 2) and on the cell surface (Tra160 and SSEA4)
- functionally assess their pluripotency
- functionally to form chimeric mice using green fluorescent protein, the GFP gene
How has the original iPS technique been adapted to improve efficiency and safety?
- using range of viruses and plasmids to deliver the genes
- safety is a consideration because many of the genes put in to make iPS genes are oncogenes
What are the main stages of iPS reprogramming?
- initiation stage
- maturation stage
- stabilisation stage
What happens in the initiation stage?
A key feature of initiation stage is MET. The starting cell population has a mesenchymal phenotype and when reprogramming is initiated they start to become more rounded and closer together. This is completed by day 6.
By day 21 they have formed colonies of pluripotent stem cells which have a very high nuclear-to-cytoplasmic ratio, and are tightly packed epithelial-like cells.
As well as MET they cells increase proliferation as a result of the cymc, lin 20 and shp 53 expression.
They also resist signals from other cells causing them to apoptose or senesce.
What are the advantages of iPS cells?
- share most of the attributes of ES cells i.e. self renewal and pluripotency
- can be generated directly from patients own cells so they are autologous and won’t be rejected
- no ethical issues
- present us with the opportunity of taking patients blood or skin cells and can reprogram then to iPSCs to study disease in test tubes
What are the disadvantages of iPS cells?
- current production methods involve inserting genes that could cause cancer
- these current methods are too inefficient to guarantee production of iP cells for each patient
- iPS cells may not differentiate as reliably to all types of cells
- great potential but need to address these challenges
What is the structure of antibodies?
Antibodies produced are a heterogenous mixture of Igs to different epitopes. They have variable regions and constant regions, light chains and heavy chains.
The variable regions vary amongst different Igs and is the specific region that ‘sticks’.
What is the normal immune response?
The normal immune response is polyclonal - involving several monoclonal lymphocytes recognising different epitopes and their progeny
What are epitopes?
part of an antigen on its surface
What are the reasons for target antigens having multiple epitopes?
- promotes a range of diverse specific antibody binding sites - not just a SINGLE antibody recognising a SINGLE antigen, will be a single antibody recognising a single epitope.
- different antibodies recognise different parts of the same antigen
What are the advantages of using antibodies as a reagent?
- high specificity
- possible avidity (how well they tick together) for virtually any antigen including novel synthetic molecules
- better than alternative - peptides need conjugation with large ‘carrier’ molecules to be immunogenic
What are considerations of the production of polyclonal antibodies?
- cost
- amount of antigen available
- volume of sera required
- phylogenic relationship between Ag origin and host
What is the process of the production of polyclonal antibodies?
1 - immunise antigen to activate B cells (add adjacent and then boost several times a few weeks apart)
2 - 4-8 weeks collect blood, clot, aliquot serum (test for activity)
3 - store (freeze) at -20C or 4C
What are the disadvantages of producing polyclonal antibodies?
- ethical issues with using animals
- every animal is different so may not be reliable for a specific test
- need to make sure the antigen being given to the animal is the same each time which is difficult if the virus is rare e.g. HIV or ebola
What is the alternative to making polyclonal antibodies?
making monoclonal antibodies
What are monoclonal antibodies?
they were first seen when looking at cancer cells. the cells are immortal so constantly produce the same antibody (as they come from the same cell). Overproduction of the same ‘monoclonal’ antibody can be dangerous, useful in diagnosis of some B lymphocyte tumours.
What are the advantages of making monoclonal antibodies?
- high specificity
- low possibility of cross reactivity with unrelated Ag
- inexhaustible supply of consistent homogenous reagent
- commercial applications
How are monoclonal antibodies produced?
1 - immunise animal with antigen of choice (test bleed reactivity for 3 weeks - antibody should be going up, then boost every day for 3 weeks)
2 - one day post booth harvest B cells
3 - take a myeloma cell (=neoplastic B cells) from a tumour culture
4 - fuse in a gel for 2/3 days then treat with a drug
[unfused B cells die - not immortal]
[unfused tumour cells die - from drug]
[fused cells are both B cells and tumour cells so they LIVE as they are immortal and resistant to the drug]
5 - plate out individual fused hybridomas and grow
6 - test each hybridoma medium supernatant for specific antibody by testing binding to antigen (ELISA)
What else can we do with monoclonal antibodies?
- can re-engineer DNa encoding heaving and light V region which can be fused to activate molecules to target activity e.g. a toxin that kills tumour cells = therapeutic usage
- can combine antigen binding site engineered in mouse with Ig constant regions form humans
What is immunoblotting (western blotting)?
separates and detects specific proteins from a protein micture
How is immunoblotting done?
1 - denature cell line by treating with denaturing reagent e.g. SDS
2 - fractionate by length of polypeptides using SDS-PAGE
3 - transfer to a robust membrane eg nitrocellulose or nylon sheet
4 - hybridise to specific labelled antibodies to see what was specific (hybridise with a specific primary antibody, was, hybridise with a secondary antibody specific for primary antibody conjugated to fluorescent label, chemo-luminescent label or dye label)
What are the used of immunoblotting?
- to determine the size (MW) of antigen
- to see how a disease is progressing
- to screen samples for antibody presence
What is immunocapture used for?
- to screen samples for antigen presence - use antibody to capture beta-HCG antigen from urine (pregnancy test)
- rapid ELISA for fluA antigen
- to screen samples for multiple antigen presence
What is immunopreciptation?
we can use this when we have a sample full of other things that we don’t want to denature (as would happen in western blotting when they were put through SDS system)
- sample labelled
- add and bind specific antibody
- add Fe binding protein A and precipitate
- wash to purify: add SDS to elute Ag from Ab
What are the uses of immunoprecipitation?
- to detect proteins of interest in a sample
- to purify native proteins of interest
- to characterise MW of proteins
- to study structure of proteins or antibody/antigen complexes
- to detect specific antibodies in a sample
What is affinity chromatography?
- purifies a soluble molecule from a mixture
- requires specific and reversible binding
What are the three stages of Affinity Chromatography?
STAGE 1: couple of ligand (antigen antibody) to a solid matrix in a chromatography column
STAGE 2: pass the sample through column allowing binding and discard the rest of the mixture
STAGE 3: elute off bound molecules. Low pH buffers disrupt non-covalent bonding
What are the advantages of Affinity Chromatography?
- reusable
- can purify late quantities (manufacture)
What is immunomagnetic separation?
The same idea as affinity chromatography except instead of beads being stuck in the column, they are magnetic, and you use a magnet to pull them to one side.
What is the process of immunomagnetic separation?
1 - mix sample and beads and bind 2 - apply magnet 3 - pour or wash supernatant 4 - resuspend purified beads 5 - elute off beads if necessary
What are the uses of immunomagnetic separation?
- mostly pulling pathogens out of sample that are very complex
- pathogen specific Ab’s allow purification and concentration from complex sample e.g. toxigenic E.Coli from faeces
What characterises epithelial cells?
They have tight junctions between the cells that restrict the passive diffusion of ions/substances across the epithelial cell layer.
What are the two oppositely facing cell surfaces?
luminal, apical, mucosal membrane: faces a hollow or fluid filled chamber
basolateral, serial membrane: normally close to a network of blood vessels
What is involved in ion transport processes in the lung epithelium?
- tight junctions
- polarised epithelium
- vectorial transport of ions (ion channels, pumps, transporters)
- transcellular and paracellular pathways for water movement
What is the driving force for Na+ entry?
the driving force is the different between the electrical and diffusional forces acting on an ion
What are the 3 potential sites for ion movement in epithelia?
1 - across the apical membrane
2 - across the basolateral membrane
3 - via the paracellular pathway
What is the equilibrium potential?
the membrane voltage (Vm) at which the driving force for net ion movement across a membrane is zero and is given by the Nernst Equation
What are methods of investigating transepithelial ion transport in whole organs?
in vivo/in vitro
water/solute transport
What are methods for investigating transepithelial ion transport in an isolated epithelium?
ussing chambers short circuit current (ISC) ion flux ratios membrane permeabilisation micro electrodes
What are methods for investigating transepitheial ion transport in membranes?
vesicles
patch clamps
artificial bilayers
What are ways of looking at water/solute?
1 - clearance methods i.e. absorption of glucose
2 - gravimetric measurement of net fluid absorption/secretion (Iv)
3 - marker infusion
What is the Ussing Technique for isolated epithelium in vitro?
1 - Short circuit current (ISC)
2 - Radiotracers
How does Short Circuit Current (ISC) work?
- the resistance of epithelia to current flow by paracellular pathways is high
- creation of a transepithelial potential (Vt)
- values for Rt and Vt can be obtained by passing a current across the epithelium and applying Ohm’s law
- the current flow required to maintain Vt at 0mV is the short circuit current (ISC)
Sum of ALL active electric ion fluxes across the epithelium
–> ion replacement, pharmacological blockade, permeabilisation studies
How do radio tracers work?
the measurement of unidirectional transepithelial radioisotope fluxes can be used to identify transport of specific ions irrespective of the transport mechanism
net fluxes across an epithelium are calculated from eh differences in oppositely directed unidirectional fluxes
ussing predicted that the unidirectional fluxes of an ion moving via simple diffusion should be of equal magnitude (Ussing flux equation)
fluxes that do not obey this equation are active transport processes
What is the simplest case where the Ussing flux ratio applies?
- identical solutions either raid elf the membrane (Ca=Cb)
- no PD i.e. short current conditions (Vt=0)
Jab/Jba = 1. exp (0)=1 — diffusion
Jab/Jba > 1 — active absorption
Jab/Jba
Why will fluxes of any single ion only represent a proportion of ISC?
Active transport correlated with ISC
If PD=0, ISC=sum of all net ion fluxes (I ionic)
What is flow cytometry?
a technique which simultaneously measures several physical characteristics belonging to a single cell in suspension. This is done by light scatter and fluorescence
What is the difference between flow cytometry and flow sorting?
flow cytometry = measuring properties of cells in flow
flow sorting = sorting (separating) cells based on properties measured in flow. Also called fluorescence-activated cell sorting (FACS)
What are methods of visualisation of antibodies?
- fluorescence microscopy
- flow cytometry
What are the basics of flow cytometry?
Cells in suspension flow in single files through an illuminated volume where they scatter light and emit fluorescence that is collected, filtered and converted to digital values that are stored on a computer.
What is fluidics? (flow cytometry)
Need to have cells in suspension flow in single file. this is accomplished by injecting the sample into a sheath fluid as it passes through a small orifice. Same fluid flows in a central core that does not mix with the sheath fluid - laminar flow. Introduction of a large volume into a small volume - hydrodynamic focussing.
About lasers…
Lasers have a single wavelength of light (or more rarely a mixture of wavelengths).
They can provide from milliwatts to watts of light.
They can be inexpensive, air-cooled units, or expensive, water-cooled units.
Lasers provide coherent light (single frequency).
What is light scatter?
When light hits a cell it is scattered in two directions. Forwards scatter is forwards and proportional to the size of the cell. At 90 degrees is side scatter, which is proportional to the granularity of the cell.
Why can we detect the four colours when we add antibodies and fluorochromes?
Because there are filters and one detector detects only light from one fluorochrome.
Emission is overlapping - PMT1 will only measure a very narrow wavelength of light, as will 2,3,4 etc
What are the electronics of flow cytometry?
- processing of signals from detectors - analogue-digital conversion
- emitted light is converted into an electronic signal by the detectors. We can capture our data and analyse it on the computer.
What is stokes shift?
the energy difference between the lowest energy peak of absorbance and the highest energy of emission
What does immunofluorescence use?
FLUOROCHROMES AND DYES
- fluorescein isothiocyanate (FITC) - GREEN
- phycoerythrin (PE) - ORANGE
- peridinin chlorophyll protein (PerCP) - RED
dozens of flourochromes on the market so lots of options about what laser and what antibodies you want to use.
Where can the single cells in suspension in flow cytometry be obtained?
- peripheral blood
- bone marrow
- fine needle aspirate
- fresh tissue
- CSF and other fluids
What are the two ways of labelling by immunofluorescence?
DIRECT: monoclonal antibodies (MoAbs) are pre conjugated to fluorochromes. This is a one-step-process.
INDIRECT: unconjugated monoclonal antibodies. The primary antibody doesn’t have the fluorochrome, but we put a secondary antibody on the antibody which does have the fluorochrome, and it binds to the primary antibody. This is a two step process. This may allow a stronger signal (more fluorochromes). This method may have to be used if you can’t buy or make an antibody with the fluorochrome on it.
What is represented by the area under the curve of a histogram?
the cells that have the particular fluorescent integrity of whatever parameter is being measured
What is gating? (flow cytometry)
A more sophisticated method. We can draw regions around the cells we know because of the side scatter on a dot plot and can make the machine only display cells in that gate on the basis of the two fluorochromes, so will be able to see the populations what whichever cell type we decide to look at.
How many populations can be identified and quantitated using 3 antibody colours?
8
with 2 we can only see 4… but if we used 4 we would see more, etc etc
What are univariate cell cycle methods? (flow cytometry)
in the simplest method, cellular DNA is detected using a fluorescent dye that binds preferentially to DNA.
Propidium iodide is most commonly used. It undergoes a dramatic increase in fluorescence upon binding DNA. It requires permeabilisation of the plasma membrane.
There are no antibodies or antigens - we are just adding something that binds to DNA. We have to permeabilise the cells to get the propidium iodide in - basically we have to punch holes in the membrane to allow it to enter.
About Propidium Iodide (PI)…
- excited at 488nm
- emits in the red
Where are the main peaks in the cell cycle under flow cytometry?
- the main peak is G0-G1
- the following smear is S phase
- there is another peak when they’re in G2M
there is sometimes a peak at the beginning showing apoptotic cells (or can be argued to just be fragments of DNA)
we can draw regions around these cells and see what proportion of the cells are in these phases
How does the assay using propidium iodide work?
- PI can not normally cross the cell membrane, so living cells will look negative on flow cytometry
- if the PI penetrates the cell membrane, it is assumed to be damaged
- cells that are brightly fluorescent with PI are damaged or dead, they will look positive
What are detection methods of apoptosis?
- by staining with PI (cells fixed)
- phosphatidyl serine, can be detected by incubating the cells with fluorescein-labelled Annexin V, and PI - i.e. using two stains together (cells not fixed)
- by staining with 7-aminoacinomycin D (cells not fixed)
How does the use of Annexin V and Propidium Iodide work in flow cytometry?
Annexin V goes to the phosphatidyl serine. In a live cell it appears totally negative because the PI can’t get in and the annexin V can’t bind as the phosphatidyl serine is all on the inside of the cell.
In early apoptosis the PS is flipped onto the outside of the membrane so annexin V can bind. However, the membrane is still in tact so PI can’t get in, so the cells will appears green but not red.
Later on in apoptosis/necrosis we lose the integrity of the membrane. The annexin can bind (green) and the PI can also get into the cell (red) so these cells will be positive for both green and red.
About 7-Aminoactinomycin D (7-AAD)…
- Excited at ~488nm
- Emits at ~660nm
- DNA specific - intercalates in G-C regions
- long emission wavelength, with FITC and PE labelled Ab for simultaneous evaluation of DNA content and 2 colour immunofluorescence using only 488nm ex.
We can label any of these things with fluorochromes and can look at different caspases at the same time. We can measure where the cell is in the apoptotic pathway.
What are the applications of flow cytometry?
- immunophenotyping of leukaemia and lymphomas
- detection of MRD
- stem cell enumeration
- CD4/CD8 in HIV
- measurement of intracellular cytokines
- study of cell cycle, viability and apoptosis
- measurement of cell proliferation
- assessment of transfection efficiency
How does cell sorting separate the droplets that we want?
When it recognises one of the droplets that fits into the category that we want the machine charges the stream so that the droplet containing the cell that we want breaks off the nozzle from the vibrations, and it is pulled into the plates and separated from the rest of the population.
What is an example of success in drug development?
LIPITOR: the biggest drug blockbuster in history with sales of $11 billion (before patient expiry)
What is an example of failure in drug development?
TORCETRAPIB: raised blood cholesterol, strong scientific basis and rationale, but failed clinical testing in 2006 after Pfizer spent $1 billion. Increased mortality rates as compared with placebo.
What are the three stages of a drug development project?
- ) DRUG DISCOVERY: candidate molecules chosen on basis of pharmacological properties
- ) PRECLINICAL DEVELOPMENT: non-human studies. Toxicity testing, pharmacokinetic analysis and formulation
- ) CLINICAL DEVELOPMENT: volunteers and patients. Efficacy testing, side-effects and potential dangers.
What are examples of drug targets?
- functional proteins
- receptors
- enzymes
- transport proteins
…not all are actually ‘draggable’ however…
What are possible non-biological limitations of drug development?
cost and complexity of drug discovery and development (regulation)
What are methods of lead finding and optimisation?
- cloning of target protein
- assay to measure functional activity
- automated systems to allow for speed and economy
- high-throughput screening of large compound libraries
- natural products, fungal, plants, bacteria
Lead optimisation, complex chemistry to increase potency, selectivity and stability
What happens in the preclinical development of drugs?
- pharmacological testing for hazardous acute effects
- preliminary toxicology testing
- pharmacokinetic testing for absorption, metabolism, distribution and elimination
- chemical and pharmaceutical development to assess feasibility of large-scale synthesis and purification as well as stability
What happens in the clinical development of drugs?
volunteers and patients, efficacy testing, side-effects and potential dangers…
PHASE I: safety and tolerability (MTD)
PHASE II: small scale efficacy/placebo controlled
PHASE III: large scale efficacy/randomised and blinded
PHASE IV: post marketing surveillance
What is avastin?
- first specific anti-angiogenesis drug
- in 2013 was the second biggest selling oncology product
- forecast to be the top selling cancer drug for 2018
- approved for colorectal, lung, kidney and ovarian cancers and eye disease
What is angiogenesis?
the formation of new blood vessels form pre-existing vessels
What are the functions of angiogenesis?
- blood vessels supply oxygen, nutrients, remove waste and allow immune surveillance
- physiological angiogenesis is essential for organ growth in the embryo and repair of wounded tissue in the adult
- abnormal genesis leads to disease –> inefficient vessel growth: stroke, MI, ulcerative disorders and neurodegeneration; excessive vessel growth: cancer, inflammatory disorders, pulmonary hypertension, blindness
What are the three types of angiogenesis?
- developmental angiogenesis/vasculogenesis
- normal angiogenesis
- pathological angiogenesis
What does developmental angiogenesis/vasculogenesis occur?
organ growth
