Techniques Flashcards
1
Q
What is the flow cytometry process?
A
- Stain sample with fluorophores conjugated to antibodies specific for target markers.
- Suspend sample in fluid and inject into the flow cytometer.
- Hydrodynamic focusing enables analysis of single cells in single droplets.
- Lasers excite fluorophores with unique excitation and emission spectra.
- Fluorophores emit light of a characteristic wavelength that can be detected.
- Light is recorded by a detector, quantifying how many cells express the fluorophore and therefore the target marker.
- Different charges are imparted based on detection, deflecting cells into different compartments.
2
Q
What is the difference between the end steps of flow cytometry with and without FACS?
A
In basic flow cytometry, forward light scatter (cell size) and side light scatter (cell granularity) are measured rather than specific cell types. Cells aren’t sorted in this analysis.
3
Q
What is the basic flow cytometry method?
A
- Wash cells in PBS.
- Stain cells for 30 minutes in the dark.
- Wash cells in PBS again.
- Suspend cells in PBS.
- Analyse in the flow cytometer.
4
Q
What are the main features of flow cytometry plots?
A
Forward scatter = cell size (lymphocytes on left, monocytes on right).
Side scatter = cell granularity (monocytes lower, granulocytes higher).
5
Q
What controls can be used for flow cytometry-related techniques?
A
VUSFIFUC: Viability dye, Unstained control, Single stain controls, Fluorophore minus one (FMO), Isotype control, Fc block control, Unstimulated vs stimulated control, Compensation controls.
6
Q
What does a viability dye control do?
A
Distinguish live from dead cells.
7
Q
What does an unstained control do?
A
Allows quantification of autofluorescence of cells, so voltage and gating can be interpreted/set appropriately.
8
Q
What does a single stained control do?
A
Allows the determination of fluorophore wavelengths, so any overlaps can be worked out and compensated for.
9
Q
What are fluorophore minus one (FMO) controls?
A
FMOs are when all Ab/fluorophores are used except one to inform gating and interpretation.
10
Q
What are isotype controls?
A
They are when antibodies against markers not on the cell type being analysed are used. Any fluorescence is due to non-specific binding.
11
Q
What are Fc block controls?
A
They are controls where Fc receptors are bound to prevent Abs from binding them, ensuring fluorescence is due to Fab binding.
12
Q
What are unstimulated vs stimulated controls?
A
Controls to see whether changes expected between stimulated and unstimulated cells are observed.
13
Q
What are compensation controls?
A
Including beads positive and negative for the target antibody, to account for fluorophore overlap.
14
Q
What are the limitations of flow cytometry?
A
- It provides no information about the locations of proteins within or on a cell.
- It is semi-quantitative.
15
Q
What should be considered when looking at flow cytometry results?
A
GAD: Is there an antibody control for each cell type?
Is there a gating strategy?
Has the data been shown quantitatively?
16
Q
What does FACS stand for?
A
Fluorescence-activated cell sorting.
17
Q
What is the use of FACS?
A
For sorting a heterogenous mixture of cells into two or more containers/populations.
18
Q
What are the advantages of FACS?
A
- Versatile as it can separate based on surface markers as well as physical characteristics.
- High throughput.
19
Q
What are the disadvantages of FACS?
A
- Slow speed, as rapid speeds would make identification difficult.
- Need a high number of starting cells with a low recovery rate (50-70%).
- Expensive, requiring highly-skilled personnel.
- Can’t sort based on intracellular antibodies as this requires permeabilisation.
20
Q
What is the use of ELISA?
A
Measurement of the amount of a soluble factor.
21
Q
What is the method for sandwich ELISA?
A
- Coat plates with capture Ab.
- Block non-specific sites.
- Incubate with sample.
- Add detection antibody coupled with horseradish peroxidase.
- Wash any non-bound sample or antibody.
- Add colorimetric substrate (TMB).
- Measure absorbance via colorimetry and use standard curves to quantify.
22
Q
What are the advantages of ELISA?
A
HHHQEV: High sensitivity, High specificity, High throughput, Easy to perform, Quantitative, Versatile.
23
Q
What are the limitations of ELISA?
A
- Lengthy and time-consuming with many steps that can go wrong.
- Temporary readouts based on reactions.
- Can only quantify the amount of substance.
- No temporal information.
24
Q
What controls are used for ELISA?
A
Positive - purified target Ag to show procedure is working.
Negative - sample not expressing target Ag.
Standard - known concentration of target Ag.
25
How is ELISA used to measure serum antibody expression?
Serial dilution. Once the range has been effectively covered, test sample and use the serial dilution curve as the standard curve.
26
What is ELISpot used for?
Measuring the frequency of cells within a population producing a cytokine of interest, and how much each cell is producing.
27
Describe the method for ELISpot.
1. Coat plates with antibody specific for the cytokine of interest.
2. Block remaining sites.
3. Plate known numbers of cells into wells.
4. Stimulate cells with antigen.
5. After 24 hours, rinse to remove cells.
6. Add a second detection mAb for cytokine coupled to horseradish peroxidase.
7. Add the colorimetric substrate.
28
How are ELISpot results analysed?
Spot number is the frequency of cytokine-producing cells.
Spot diameter is proportional to the amount of cytokine produced.
29
What are the controls for ELISpot?
Positive - stimulation known to induce cytokine secretion.
Negative - unstimulated cells.
Background - no cells but all other reagents.
30
How may ELISA and ELISpot be used?
ELISA to determine that a factor is present and its concentration.
ELISpot to quantify the number of cells producing the factor in tissue samples.
31
What is intracellular cytokine staining used for?
To study cytokine production in individuals using flow cytometry.
32
What information does intracellular cytokine staining provide?
Information about the cellular sources of cytokine and proportions of different populations producing cytokine.
33
Describe the method of intracellular cytokine staining.
1. Cells are stimulated.
2. Brefeldin A is added to prevent secretion of de novo synthesis of cytokines.
3. Stain cells for surface markers.
4. Permeabilise cells using detergent to enable entry of cytokine-specific mAbs conjugated to fluorophores.
5. Analyse via flow cytometry.
34
What are the limitations of intracellular cytokine staining?
1. No temporal information - only provides a 'snapshot'.
2. Semi-quantitative - cannot relate medium fluorescence intensity to the known quantity of cytokine.
35
What is RNA-seq used for?
Rapid profiling and investigation of the transcriptome.
36
What is an alternative to RNA-seq?
Microarrays.
37
What does RNA-seq show?
The cellular content of all RNAs.
38
Describe the method of RNA-seq.
1. Cells are lysed either physically or using a reagent.
2. RNA is isolated via acid guanidinium thiocyanate-phenol-chloroform extraction.
3. Separate the solvents into 2 phases - upper aqueous phase containing nucleic acids and lower phase containing proteins and lipids.
4. Reverse transcribe RNA to form a cDNA library.
5. Sequence this library via next generation sequencing.
39
What are the advantages of RNA-seq?
PRADSP: Previous sequence information is not required, Range is highly dynamic, Alternative splicing detected, Direct sequence alignment, SNPs can be identified, Paralogous genes can be defined.
40
What are the disadvantages of RNA-seq?
1. Process not fully optimised.
2. High setup and ongoing cost.
3. Requires high power computing facilities.
4. Analysis of splice variants and paralogues is complex.
41
What are the 3 advantages of microarrays?
1. Well-defined protocols for hybridisation.
2. Well-defined analysis pipelines.
3. Relatively low cost.
42
What are the disadvantages of microarrays?
1. Pre-defined sequences only can be analysed.
2. Dynamic range limited by scanner.
3. Relies on hybridisation which may be non-specific.
4. Generally won't identify splice variants.
5. High variance for low expressed genes.
6. Might not give paralogue information.
43
Which of RNA-seq and microarrays have better sensitivity and specificity?
RNA-seq is better for both.
44
What can cause an issue for RNA-based sequencing methods?
Lack of poly(A) tails can lead to missing non-polyadenylated sequences.
45
What is the gold standard to define cell type and phenotype?
Single cell RNA-seq (scRNA-seq).
46
What is the use of scRNA-seq?
Analysis of the transcriptome of a single cell.
47
What is the conceptual advantage of sc vs normal RNA seq?
sc sequencing allows single cells within a population to be analysed, revealing heterogeneity.
48
Describe the method of scRNA-seq.
1. Isolate a single cell.
2. Isolate its RNA as in standard RNA-seq.
3. Reverse transcribe into cDNA.
4. Amplify the cDNA.
5. Sequence the cDNA.
6. Compare sequences to a library to identify the cell.
49
What are the advantages of scRNA-seq?
1. Gold standard to identify and define cell type and phenotype.
2. Characterises heterogeneity within cellular populations.
50
What are the disadvantages of scRNA-seq?
1. May miss non-polyadenylated sequences.
2. Bias of transcript coverage.
3. Data can be over or under-clustered.
4. Gating on clustering can be subjective.
5. Clusters can be caused by biological and technical variation.
51
What is the use of cytometric bead arrays?
Measurement of a variety of soluble and intracellular proteins.
52
Describe the method of cytometric bead arrays.
1. Fluorescent beads are pre-coated with a capture mAb specific for a protein.
2. Sample is incubated with beads to allow protein binding.
3. Protein-specific mAbs labelled with different fluorophores added.
4. Samples are analysed with FACS.
53
What is the advantage of cytometric bead arrays?
Broader dynamic range compared to ELISA.
54
What is Western blot also known as?
Immunoblotting.
55
What is the use of Western Blots?
Detection of the presence of specific proteins.
56
Describe the method for performing Western blots.
1. Separate molecules via gel electrophoresis.
2. Transfer molecules onto a matrix.
3. Block non-specific sites.
4. Add a primary antibody that binds protein.
5. Add a secondary antibody that binds the primary antibody.
6. This secondary Ab is complexed to an enzyme, so when substrate is added a color change reaction occurs.
57
What are the advantages of Western blots?
1. High sensitivity.
2. High specificity.
58
What are the limitations of Western blots?
1. Semi-quantitative.
2. Prone to false results.
3. Expensive.
4. Requires specific primary Abs.
59
Name the 4 types of control for Western blots.
Positive, Negative, Endogenous, Loading.
60
What is the positive control for Western blots?
Lysate from a cell line or tissue known to express the protein of interest.
61
What is the negative control for Western blots?
Lysate from a sample known to not express the target protein.
62
What is the endogenous control for Western blots?
An endogenous lysate known to express the target protein, needed when examining recombinant proteins.
63
What is the loading control in Western blots?
Addition of a different protein (e.g. beta-actin) for semi-quantification of results.
64
How may Western blots be made quantitative?
Using densitometry to measure band density optically against the values for the loading control.
65
What are Northern blots used for?
Detection of RNA.
66
What are Southern blots used for?
Detection of DNA.
67
Describe the method of Northern blots.
1. Extract RNA from tissue.
2. Separate RNA via gel electrophoresis.
3. Transfer RNA samples to a nylon membrane.
4. Add fluorescent probes that bind mRNA of interest.
5. Image using X-ray film.
68
What are the advantages of Northern blots?
1. Detects small changes in gene expression.
2. Detects alternate splice products.
3. Can store membranes for years.
69
What are the disadvantages of Northern blots?
1. Samples can be degraded by RNases.
2. Can only examine one gene at a time.
70
What is Cre/Lox?
A site-specific recombinase technology used to modify DNA.
71
What can CreLox do that germline knockouts cannot?
Site- and time-specific knockout of genes.
72
What flanks the target gene in Cre/Lox?
loxP sites.
73
What are the 3 types of modification/placements in Cre/Lox?
1. Excision - cis placement of loxP sites in the same directional orientation.
2. Inversion - cis placement of loxP sites in the opposite directional orientation.
3. Translocation - trans placement of loxP sites.
74
How may spatiotemporal specificity be conferred for Cre/Lox?
Using Cre fused to the oestrogen receptor (Cre-ER) - activate the receptor using tamoxifen.
## Footnote
It is limited as Cre-ER becomes leaky over time - the receptor will migrate to the nucleus in the absence of tamoxifen.
75
What are the 5 limitations of Cre/Lox?
CLUMP, Cre phenotype, Low breeding efficiency, Unexpected expression, Mosaicism, Prior expression.
76
How does mosaicism limit Cre/Lox?
Some cells may not express Cre, and some LoxP may not recombine.
77
How may unexpected expression be mitigated in Cre/Lox?
Using Cre-positive males for breeding, test expression with a reporter.
78
Why is breeding efficiency low in Cre/Lox mice?
Can require recombination events for optimal performance.
When making deletions, Cre transgene should be bred out to reduce phenotypic interference.
79
What may Cre phenotypes be, and what can cause them?
Phenotype may be an alteration in gene expression of the entered gene, or Cre toxicity.
Can be caused by gene insertion effects and actions of strong promoters.
80
Why may early gene expression hamper Cre/Lox studies?
If the gene has fulfilled its function already, then studies looking at its modification are useless.
81
What are the main controls used for Cre/Lox studies?
Just LoxP, Just Cre, Wild-type.
82
What is the polymerase chain reaction used for?
DNA amplification.
83
Describe the method of PCR.
Add reagents and template to PCR tubes.
Mix and centrifuge.
Amplify using a thermo cycler.
Evaluate amplified DNA using gel electrophoresis followed by staining and sequencing.
84
What are the reagents used for PCR?
Taq DNA polymerase,
PCR grade water,
DNA primers (forward and reverse),
Oligonucleotides,
DNA to be amplified, dNTPs.
85
What are the controls for PCR and what do they do?
Positive - control reaction with a known amount of template - checks the primer-probe set and reaction works.
Negative - control reaction with everything bar the template to allow contaminant detection.
86
What are the 3 main advantages of PCR?
High sensitivity,
Can test for antimicrobial resistance,
Quick.
87
What are the 2 disadvantages of PCR?
Lower specificity,
Sensitive to cross contamination.
88
What is the use of qPCR?
To quantify the amount of DNA.
89
How does qPCR work (differences in the PCR step)?
In the annealing stage, probe and primers anneal to the DNA target.
Taq polymerase extends DNA from the primer.
When Taq polymerase reaches the probe, the exonuclease cuts the probe which separates the fluorescent reporter.
This produces an increase in fluorescence which is detected.
90
What are the 4 advantages of qPCR?
Cost-effective,
Time-efficient,
Sensitive,
Specific.
91
What is RT-PCR used for?
To obtain DNA from RNA.
92
What is the 1 step method of RT-PCR? What are the 3 main advantages of this?
RNA template is reverse transcribed and the cDNA is amplified in one tube/step, by including both reverse transcriptase and DNA polymerase in the same tube.
This is simple, fast, and has less risk for contamination.
93
What is the 2 step method for RT-PCR? What are the main advantages of this?
RNA template is reverse transcribed, then cDNA is amplified in a separate reaction.
This is more sensitive than the 1 step method, and generates cDNA for other uses.
94
What are the 6 main advantages of RT-PCR?
Both quantitative and qualitative,
Easy to use,
Rapid,
Cost effective,
High sensitivity,
High specificity.
95
What are the 2 main disadvantages of RT-PCR?
So sensitive that it is very susceptible to contamination,
Needs high quality RNA for good results.
96
What are microarrays used for?
Detection of the expression of thousands of genes simultaneously.
97
What is a microarray (the object)?
A microscope slide printed with spots in defined positions, each containing a known DNA sequence of a gene (each spot/sequence is a chip).
98
Describe the method for conducting microarrays.
Collect mRNA from an experimental sample (disease state) and a healthy reference,
Convert into cDNA and label each sample with a different fluorescent probe,
Mix samples and allow to bind to the microarray slide,
cDNA and DNA hybridise,
Scan to measure expression, can see no expression, differential expression, and simultaneous expression.
99
What are the 3 main limitations of microarrays?
Thresholds for significant differences is largely arbitrary,
Sample heterogeneity can make significance invalid,
Doesn't show whether altered expression is due to population differences or a critical cellular disease event.
100
What is surface plasmon resonance (SPR) used for?
Measuring the affinity of an interaction between two molecules (usually receptor and ligand).
101
Describe the method for conducting SPR.
Immobilise protein on gold plated surface,
Allow soluble molecules to flow over the surface, bind to protein, and reach equilibrium,
Binding changes the resonance of the protein as properties change,
On/off rate shows strength and duration of interaction to study dissociation constants.
102
What are the 5 main advantages of SPR?
Real time measurement,
Fast,
Can be done on small samples,
Highly sensitive and specific,
Doesn't require standards.
103
What are the 5 main disadvantages of SPR?
Common data misinterpretations,
Control experiments require care in designing, Expensive,
Affected by steric hindrance,
Inaccurate for weaker interactions where Kd is greater than 100 micromolars.
104
What is the use of luciferase reporter assays?
They are used to study gene regulation at the level of transcription.
105
Describe the method for conducting luciferase reporter assays.
Transduce cells with a DNA plasmid expressing a construct designed to alter transcription and a luciferase reporter gene,
Culture cells for a few days,
Lyse cells,
Add luciferase substrate,
Measure bioluminescence - more light with higher luciferase expression.
106
What is a luciferase reporter gene and what does it do?
A DNA plasmid with its transcription regulatory element (TRE) fused to DNA encoding luciferase, so luciferase is produced when the TRE is activated.
107
What are the 4 main advantages of luciferase reporter assays?
Sensitive,
Reproducible,
Compatible with other control reporters,
Reagents are not radioactive so less damaging to cells.
108
What are the 2 main disadvantages of luciferase reporter assays?
Requires exogenous substrates, 4-6 hour delay from stimulation to response to allow transcription to occur.
109
What does immunoprecipitation do?
Uses antibodies to precipitate a target protein out of solution - isolates and concentrates it.
110
Describe the method used to conduct immunoprecipitation assays.
Lyse cells,
Add antibodies specific for the target protein to cell lysate,
Add beads coated with protein A or G to make Ab-protein complexes insoluble,
Pellet out complexes via centrifugation.
111
What are the controls used in immunoprecipitation assays?
Positive - purified recombined target protein,
Negative - cells or tissues in which the target protein isn't expressed/empty beads combined with cell lysate without Ab/Ab without beads.
112
What are the 2 main advantages of immunoprecipitation?
Easy, Fast.
113
What does coimmunoprecipitation do and what is it used for?
Immunoprecipitates intact protein complexes, used to identify interactions between proteins by precipitating a known protein, and pulling out any proteins it is bound to.
114
Describe the method for conducting coimmunoprecipitation assays.
Prepare cell lysate,
Add an antibody specific for the target protein,
Use resin/Sepharose/beads conjugated with IgG binding protein A or G,
Wash to remove non-complexed proteins,
Analyse bound proteins by electrophoresis and immunoblotting or mass spectrometry.
115
What are the 4 main advantages of coimmunoprecipitation?
Easy, Fast,
Compatible with most protein analysis techniques,
Native and bait proteins stay in native conformation.
116
What are the 3 main disadvantages of coimmunoprecipitation?
Can miss low affinity or transient protein interactions,
May miss indirect interactions e.g. ones where another protein provides structural support,
Non-specific binding could produce false positives.
117
What are chromatin immunoprecipitation (ChIP) assays used for?
Used to identify DNA binding sites on the genome for a protein of interest.
118
Describe the method used for conducting ChIP assays.
Cross-link proteins bound to DNA using formaldehyde,
Shear the DNA and lyse cells,
Conduct IP, will also pull out the cross-linked DNA,
Remove, purify, and analyse DNA.
119
What are the 2 main advantages of ChIP?
It can be used to study non-histone proteins,
It doesn't require a large number of cells.
120
What are the 5 main limitations of ChIP?
Can be hard to access proteins,
Expensive,
Precipitation is often inefficient,
Cross-linking may fix minor, irrelevant interactions,
Cannot determine if protein directly interacts with DNA or is part of a greater macromolecular complex.
121
What is the use of cytotoxicity assays?
They are used to assess target cell killing by cells.
122
Describe the method for the conduction of cytotoxicity assays.
Obtain effector and target cells (specific - if looking at HIV cell killing need HIV protein target cells),
Co-culture cells (can use different target:effector ratios to determine dose-dependency),
Determine cytotoxicity using various methods.
123
What are the methods that may be used to determine cytotoxicity in the eponymous array?
Flow cytometry and radioactive chromatin release.
124
How may flow cytometry be used to determine cytotoxicity?
Using fluorescent dyes that penetrate dead cells,
Using antibodies that detect caspase activation,
Using antibodies for molecules upregulated on cell death such as FasL.
125
How is the radioactive chromatin release assay used to detect cytotoxicity in the eponymous assay?
The target cell is incubated with 51Cr which is internalised, and then released into the supernatant upon cell lysis.
Separate the supernatant from cells then detect radioactivity with a Geiger counter.
126
What are tetramer assays used for?
Detection and quantification of antigen-specific T cells.
127
What are tetramers in the eponymous assay?
4 MHC molecules bound to a specific peptide epitope derived from the antigen of interest.
128
Describe the method used for the conduction of tetramer assays.
MHC molecules are biotinylated, causing them to assemble into a tetrameric complex when mixed with streptavidin.
Tetramers are conjugated into fluorophores so can be detected with flow cytometry.
Tetramers bind cognate TCR on cells, so can be sorted with flow cytometry (alone the MHC molecule is too low to bind the TCR).
129
What is the negative control in tetramer assays? What does it do?
A peptide known to not react with the studied sample/system - determines background staining.
130
What are proliferation assays + replicate tracking used for? How?
Measuring cell proliferation - fluorescent dye is split between daughter cells when a cell divides so fluorescence intensity decreases with increased proliferation.
131
What 2 dyes are commonly used in proliferation assays/replicate tracking and what do they do?
CarboxyFluoroscein Succinimidyl Ester (CFSE) - binds intracellular lysine residues and free amines;
Celltrace violet binds free amines on the surface and inside of cells.
132
Describe the method used for the conduction of proliferation assays + replicate tracking.
Stain cells,
Stimulate cells,
Measure fluorescence.
133
What are the assumptions when using dyes for proliferation assays + replicate tracking? Consequently, which dye is better to use?
The dye is stable in vivo,
The cell is non-toxic, and doesn't alter cell physiology (CellTrace Violet better for this).
134
What is transfection/transduction?
The artificial introduction of material such as DNA, RNA, or proteins into cells resulting in transient or sustained expression of a protein of interest/activity of a molecule within a cell.
135
What is the difference between transfection and transduction?
Transfection uses viral methods, transduction doesn't.
136
What are the 2 main vehicles for transfection?
Adeno-associated virus (AAV) and lentivirus.
137
What is transfection like using AAV?
DNA exists as episomes rather than being incorporated into the genome.
Expression persists a few years.
138
What is transfection like using lentivirus?
DNA is incorporated in a stochastic manner, sustaining expression for the cell's lifespan.
139
What are the 6 main advantages of lentivirus transfection?
Infects a variety of cells,
High transfection efficiency,
Doesn't affect normal cellular function,
Minimal immunogenicity,
Rapid and long-term expression,
Compatible with multiple transcriptional promoters.
140
What are the 3 main disadvantages of lentivirus transfection?
Non-specific insertion of transgene (low/no expression with weak/silent promoter, regulation different than at the natural locus),
Limited to inserts of 10kb or less, Risk of insertional mutagenesis.
141
What are the 3 main non-viral methods of transduction?
Electroporation,
Microinjection,
Lipofection.
142
What are the controls for transfection/transduction assays?
Positive - test for efficacy with a known insert like galactosidase,
Negative - non-transfected control.
143
What are the 6 main advantages to using animal models?
Can use larger sample sizes so more power,
Fewer ethical considerations so can be more invasive,
Accelerated development so faster time course for experiments.
144
What are inserts of 10kb or less associated with?
Risk of insertional mutagenesis
145
What are the 3 main non-viral methods of transduction?
Electroporation,
Microinjection,
Lipofection
146
What are the controls for transfection/transduction assays?
Positive - test for efficacy with a known insert like galactosidase;
Negative - non-transfected control.
147
What are the 6 main advantages to using animal models?
1. Can use larger sample sizes so more power
2. Fewer ethical considerations so can be more invasive
3. Accelerated development so faster time course for experiments
4. Rear from birth so can control upbringing
5. Cheaper
6. Inbred animals are genetically similar so less variance in responses
148
What are the 4 main limitations of animal models?
1. Inbred animals may have pathogenic recessive alleles
2. Physiological and anatomical differences may limit appropriateness
3. Accelerated development reduces follow-up
4. Hard to know whether proxy measures of behaviour are appropriate
149
What are the 2 main advantages of mouse models of flu?
1. Many different strains to assess the genetic effects of mice on response
2. Genetic manipulation can assess pathways
150
What are the 5 main disadvantages of mouse flu models?
1. Flu is not a natural pathogen of mice
2. Surface receptor distribution may be different so differential cellular infection
3. Mainly use attenuated strains that don't reflect pathogenicity
4. Recombinant virus required to track immune response
5. Therapeutics may not translate
151
What are congenic mice?
Inbred mice with the same genetic material bar one area (larger than a locus) of a chromosome
152
What is the most commonly-used chimeric mouse and what is it used to do?
CD45.1/CD45.2 congenic mouse (BL6 background).
mAbs differentially bind these alleles so can distinguish cells using flow cytometry.
Used to assess the immune responses of 2 cohorts in 1 host which minimises variability
153
What are the 2 main disadvantages of CD45.1/.2 chimeric mice?
1. Irradiation can introduce artefacts
2. Mice require long-term treatment with antibiotics
154
How are TCR transgenic mice developed?
1. Desired epitope is identified and specific T cells isolated
2. Sequence TCR DNA
3. Generate cDNA
4. Inject cDNA into fertilised mouse embryo
5. Implant embryo into a pseudopregnant female
6. Breed offspring with RAG-KO mouse (these produce no T cells)
155
What are the 2 main advantages of TCR transgenic mice?
1. Monoclonal cells of a known specificity
2. TCR expression is normal
156
What are the 2 main disadvantages of TCR transgenic mice?
1. Disrupted thymic architecture
2. Higher specific TCR frequency so more competition, leading to weak and abnormal responses
157
How may some of the disadvantages to transgenic TCR mice be overcome?
Transfering transgenic TCR cells into recipients
158
What is the advantage of transgenic TCR transfer?
Can control the number of antigen-specific T cells delivered to a naive mouse
159
What is the disadvantage of transferring transgenic TCR cells?
Cells must be followed in mice, and natural variation leads to variability so need a larger sample size
160
What are fluorescent proteins used for?
Labelling of a specific molecule in a cell or tissue
161
What are the various uses of fluorescent protein labelling?
1. Marking cells of a specific phenotype
2. Co-expressing with a protein to see which cells express the protein
3. Conjugation to protein to visualise its trafficking within cell
162
What are the 2 main advantages to fluorescent protein labelling?
1. Can image live cells
2. Very versatile
163
What are the 2 main disadvantages of fluorescent protein labelling?
1. Expression can alter physiology
2. Expression takes hours to days
164
How can GFP alter physiology?
1. It is immunogenic in mouse so can cause inflammation
2. Tagging proteins with GFP generally stabilises them, so are degraded less rapidly than endogenous protein
3. GFP can sterically block protein interactions with substrates
165
What are calcium flux assays used for?
Detecting intracellular Ca2+ mobilisation in cells
166
Name 3 calcium-binding dyes.
Fluo-8, Fura-2, Indo1
167
How do calcium binding dyes work?
They are conjugated to carboxyl groups masked as acetyoxymethyl esters, which make the molecule lipophilic so can enter cells.
Esterases inside cells free the carboxyl groups so the dye can bind free Ca2+.
Binding alters fluorescent properties.
Ratio of bound:unbound dye can be quantified via flow cytometry.
168
Describe the method used for the conduction of calcium flux assays.
1. Add dye to the cell solution
2. Incubate to preload
3. Stimulate (do what you want to measure calcium flux in response to)
4. Measure [Ca2+] by flow cytometry using dye-specific wavelengths
169
What are myeloperoxidase (MPO) assays used for? How?
Measuring neutrophil recruitment - measures the amount of MPO which is produced by neutrophils
170
Describe the method used for the conduction of MPO assays.
1. Homogenise tissue
2. Add chloride, bromide, and thiocyanate (MPO substrates)
3. Add TNB probe
4. Measure absorbance
5. Compare with standard curve and normalise to total protein content
171
What are the 3 main limitations of MPO assays?
1. Assumes processing doesn't affect catalytic activity of MPO
2. Assumes MPO is only expressed by PMNs
3. Assumes expression in PMNs doesn't change between experiments
172
What are radioimmunoassays used for?
Quantifying antigen concentrations in the blood
173
Describe the method used to conduct radioimmunoassays.
1. Add a known amount of the investigated antigen bound to a radioactive element
2. Mix with a known amount of antibody against that antigen, which binds
3. Add a sample containing the antigen of interest, which competes with the radioactive antigen
4. Compare the ratio of radioactivity present in the supernatant to standards to determine antigen presence in the sample
174
What is the main advantage of radioimmunoassays?
Has an extremely high sensitivity
175
What are the 3 main disadvantages of radioimmunoassays?
1. Expensive
2. Radioactive compounds have a short shelf-life
3. Disposal of radioactive waste is difficult
176
What is mass spectrometry used for?
Identification of a huge range of molecules
177
What analytical method is often used upstream of mass spectrometry, and why?
Liquid chromatography to separate compounds in the sample
178
What are the 4 main advantages of mass spectrometry?
1. Rapid
2. Sensitive
3. Qualitative
4. Quantitative
179
What is the principle that peptide mass fingerprinting is based on?
That enzymatic digestion of a protein will produce a unique peptide fingerprint, which can be identified and cross-referenced with a database for protein identification
180
Describe the method used to conduct peptide mass fingerprinting assays.
1. Cells are lysed
2. Lysates are centrifuged, incubated with biotinylated protein, and injected with resin
3. Bound precipitates eluted and trypsin digested to generate peptides
4. Peptides separated by liquid chromatography and injected into mass spectrometer
5. Peptides are analysed with mass spec generating a mass-charge (m/z) spectrum
6. Compared to a reference spectrum to identify peptides
7. Peptide fingerprint referenced against a database
181
What are the 2 main advantages of peptide mass fingerprinting?
Sensitive and accesses proteins easily
182
What may cause false positives in peptide mass fingerprinting?
1. Incubation of sample with lysates with different intracellular proteins
2. Indirect binding patterns
3. Errors in protein identification algorithms
183
What may cause false negatives in peptide mass fingerprinting?
1. Weak binding
2. Poor peptide ionisation
3. Binding proteins not being expressed on the cells used
184
What is TUNEL staining?
A staining method that detects DNA breaks formed during the final phase of apoptosis, when DNA fragmentation takes place
185
What are morpholinos?
18-25 nucleotide long oligomers that block access of other molecules to specific RNA sequences, to knock down gene function
186
What is forward genetics?
Working out what gene is responsible for a change in phenotype
187
What is reverse genetics?
Working out the function of the gene through knock-ins/knock-outs and assessing the change in function
188
What is peptide pulsing?
Incubation of a cell (usually an immune cell) with peptides, at a high concentration to cause MHC binding
189
What are the advantages and disadvantages of optogenetics?
Advantages - high temporal resolution (microseconds), anatomical control (even to subcellular compartments), can change effect with wavelength;
Disadvantages - very invasive, costly, can be activated by heat if long wavelength, can be phototoxic if short wavelength
190
What are the advantages and disadvantages of DREADDs?
Advantages - can be activated on a longer timeframe, non-restrictive, non-invasive;
Disadvantages - low temporal resolution, low spatial resolution, lack of diversity, potentially constitutive activation
191
What is the difference between RT-PCR and RNA-seq?
RT-PCR reports expression of one gene at a certain time while RNA-seq (and microarray hybridisation) report expression of all genes at once
