Practical 2: Flow Cytometry Flashcards
Define flow cytometry
A technique for counting, examining and sorting microscopic particles suspended in a stream of fluid
How does the flow cytometer work
A laser beam directed at a hydro-drynamically focused stream of fluid containing cells in single-file excites cells as they move by
A number of detectors pick measure properties such as forward scatter, side scatter and fluorescence at different wavelengths
The protein expression of cells can be measured by incubating cells with fluorescently labelled antibodies
How are fluorescently labelled antibodies used in flow cytometry?
The cell suspension is insubated with labelled antibody(s)
The cells are then aspirated from the sample tubes and hydrodynamically forced in single file by a laser
By combining antibodies with different fluorescent labels that emit light at different wavelengths we can carry out high throughout, multi-paramater analysis
The emitted light is detected by an optical system where specialised software on an adjoined computer can export the data as either dot plots or histograms
What is meant by hydrodrynamic focussing
Cells in sheath fluid flow through the tubing in a laminar fasion, as the tubing diameter narrows the cells are forced into single life before the laser
i.e. sheath fluid runs along either side of sample - so that sample is in laminar flow
Narrowing of tubing, along with work of sheath fluid, focuses the cells into single file
What are the two most common fluorophores using in flow cytometry?
Fluorescein (FITC)
R-phycoerythrin (PE)
What is spectral overlap?
One must be careful when deciding on which fluorescent label to use in order to avoid too much spectral overlap
This is where the light emitted by the fluorophores is of a similar wacelength and thus positive cell populations will bleed into each other e.g. there is significant overlap between FITC and PE
What does forward and side scatter measure?
Forward scatter - FSC
Side scatter - SSC
What is gating?
Gating is used to focus on a population of interest
- a method to define cell population of interest
- typically performed using forward and sde scatter
Within the lymphocyte gate what would CD3- CD56+ cells be?
NK cells
How many lasers are there in most flow cytometers?
There is usually 2, which can excit cells at different wavelengths
Three or more lasers can be used in larger machines
What does the light from the laser do?
The light interrogates cells as they pass by
NB: it is the light from the laser that does this not the laser itself
How are fluorophores used?
Light from a laser is absorbed by the fluorphores
The fluorophores cannot hold onto this light so it emits it back out
The light emitted is of a different wavelength to the light absorbed
This light is then detected by a detecter
How can flow cytometry be used on tissue?
A hydrodynamically focused beam of cells is required for flow cytometry
you cannot force solid material through the cytometer therfore tissue must be disaggregated/broken down
Enzymes are used to do this, a cell suspension is made
Need to be careful what enzyme you use though as you dont want it to target the antigen you are looking to detect in flow
Need to avoid weakening of antigen expression while disaggregating the tissue enough to make a cell suspension
What kind of controls do you use for flow?
You want to use negaive cells e.g. double negative cells, cells which contains neither signals you want to identify
How are mirrors used in flow cytometry?
Mirrors are used to change direction of light, it doesnt alter the wavelength of emitted light in anyway but they are a useful way of making our analysers small then they have to be, can make them a lot more compact through the use of mirrors
What is spectral overlap compensation?
The mathematical process by which we correct multiparameter flow cytometric data for spectral overlap
This overlap results from the use of fluorescent dyes that are measurable in more than one detector
This is the use of a mathematical algorithm to remove any “spilover” between two different fluorophore spectra
List the major advantages of Flow Cytometry
Generatioin of correlative information about single cells within a heterogenous sample preparation in a high-throughput fashion
Rapid a nd quanititative measurement of up to 30 parameters of cell phenotype simultaneously in a highly sensitive and reproducible manner
High speicificty for discrete cell subsets and rare populations such as IL-17A, IL-22 and IFN-y producing CD4+ cells in untreated psoriasis
Rare cell subsets with frequencies as low as 0.01% can be detected
Flow cytometric cell sorting also allows for the isolation (and susbequent investigation) of cells of interest to very high purity
What are the major limitations of flow cytometry?
Cells must be in a single-cell suspensioni to be evaluated -> problem for tissue as it must be disrupter which can affect antigens
The number of paramters per cell that can be mesured is limited by the number of detectors to less than two dozen but due to the spillover effect only 6-12 color experiments are routinely performed
There is a lack of standardisation on how flow data is analysed and reported
Flow data can become very complicated requiring very specialised human experts
Give an example of a dye used in flow cytometry
Propidium iodide
How is propidium iodide used in flow cytometry?
It binds to phosphodidyl serine which is usually held inside cells
Binding will only occor if opoptotic cells are present i.e. if cell contents are available for binding
How is propidium iodide used in flow cytometry?
It binds to phosphodidyl serine which is usually held inside cells
Binding will only occor if opoptotic cells are present i.e. if cell contents are available for binding
What is Jurkat’s Cell line?
immortal cell line of CD8+ cells
What does Annexin V-FITC bind
apoptotic cells
What would low propidium iodide binding but positive annexin-V binding indicate vs high PI and A-V bining
+ A-V but low PI indicates aptotic cell but only early stage
+ A-V and high PI indicates aptotic cell in late stage - necrotic
Your PBMC suspension has a final volume of 3mls with a concentration of 5.78x10^6/ml
What is the total no. of cells you have isolated from your anti-coagulated peripheral blood sample?
Multiply concentration by volume
- 5.78x10^6/ml x3 = 17.34 cells in 3mls PBMC sol
How would you work out how much diluent (PBS) you should add to have a concentration of 1x10^6/ml?
Concentration: 5.78x10^6/ml
Volume: 3mls
C1V1 = C2V2
(5.78)(3)=(1)(V2)
(17.34)/(1)= V2
17.34 = V2
17.34 -3.0mls (in tube already) = 14.34mls buffer to be added to your original 3mls of solution to get a concentration of 1x10^6/ml
With a 1x10^6 cells/ml PBMC suspension, what volume is required so tht 1x10^5 PBMCs are added to each FC tube for analysis?
1x10^6 cells/ml = 1x10^6 cells/1000ul
1x10^5 cells/ml = 1x10^5 cels/100ul
100ul = 1ml => 1ml is needed for 1x10^5 cells/ml
List five specific examples of applications of flow cytometry
Immunophenotyping
Levels of cytokine production
Measurement of cell proliferation
Cell sorting
Solid organn transplantation
How can flow be used in immunophenotyping?
CD4+ (helper) cells vs CD8+ cytotoxic cells in HIV analysis
Gate drawn on lymphocytes - CD8, CD4, Bcells, NKs
Talk about dual analysis used in FCXM
We look for two signals:
- Signal 1 = what specific HLA type coated bead the antibody in patient serum has bound to i.e. what anti-HLA
- Signal 2 = anti-C1q FITC conjugated antibody - only get signal if C1q binds - is the recipient antibody complement activator or not
List some specific examples of flow cytometry uses
HIV analysis
Immunodeficiency in LAD1
XLA
Basophil activation in allergy
Talk about flow for LAD1
LAD1 = Leukocyte adhesion deficiency-1
- inability of leukocytes to migrate to site of infection (B and T cells affected)
Mutations in CD18 gene resulting in defective or dificient B2 chain of B2 integrins
Characterised by flow through a lack of CD18 positivity in PBMC
Talk about flow for XLA
X-linked agammaglobublinaemia
Characterised by a lack of CD19 positive B cells
mutations in BTK gene - absence of BTK+ B cells
- BTK -/ve and CD20 -/ve
Healthy person will have CD20+ BTK+ cells
Talk about flow cytometry for basophil activation in allergic reactions
Basophils upregulate CD63 and CD203c when activated
Increase in CD63 and Cd203 positivity -> degree of positivity increased
How can we investigate intracellular cytokine production?
By permeabilising cells with saponin we can determine which cell populations are producing a cytokine of interest
- activated T cells are treated with an inhibitor which blocks protein export allowing cytokines to accumulate in the ER
- the cell is fixed and permeabilised with mild detergents
- cytokine-specific antibodies penetrate the cell to bind thhe intracellular cytokine molecules
Give two sepcific examples of flow cytometry looking at intracellular cytokine production
Analysis of IL-10 production in both unstimuated and stimulated helper T cells
- increase in IL-10 positive T cells when stimulated
CD3+ cells stained for intracellular cytokine expression in response to M. tuberculosis exposure
- Increase in IFN-y producing cells
What is CFSE
Carboxyfluorescein diacetate succinimidyl ester
Is is a blue laser excitable dye that is used for flow cytometric monitoring of cell divisions
Talk about the use of CFSE in flow cytometry for cell proliferation
(4)
CFSE dye passively diffuses across cell membranes and is cleaved by intracellular esterases within viable cells
The cleaved dye becomes highly fluorescent and covalently binds to protein amine groups within the cells via its succinimidyl ester group
Non viable cells remain nonfluorescent
As viable cells divide the CFSE dye is distributed uniformly between daughter cells, each retaining half the intensity of the parent cell
Used to monitor distinct generations of proliferating cells through dye dilution with each generation of cells appearing as a different peak on a histogram
Talk about flow cytometry as a means of cell sorting
Cells in suspension are taged with fluorescenct matkers specific to target cell
Labelled cells are sent under pressure through a small nozzle and pass through an electric field
A laser beam passes through one cell
A cell generates a negative charge if it fluoresces and a positive charge if it does not
Positive cells are separated into one container and negative cells are separated into another
What are some post-transplant applications of flow cytometry?
Ab mediated rejection (AMR) diagnosis
Graft prognosis
Therapeutic monitoring
Talk about flow for AMR diagnosis
Detection of de novo DSA
- earlier detection the better the surival of the organ
- treatment includes plasmaapheresis, IVIG and anti-CD20 therapies
- 30% of renal transplant patients develop DSA and 66% of these experience acute rejection
Talk about flow for AMR diagnosis
Detection of de novo DSA
- earlier detection the better the surival of the organ
- treatment includes plasmaapheresis, IVIG and anti-CD20 therapies
- 30% of renal transplant patients develop DSA and 66% of these experience acute rejection
What four ways is flow used for graft prognosis?
CMV monitoring
- CMV antigen - speicific T cells (CASTs) cytokine production (LOOK UP)
EBV reactivation monitoring
- causes PTLD - massive proliferation of B cells which can kill transplant patients
Monitoring regulatory cells
- monitoring Tregs and Mregs
- Cd8+, CD28- Treg measurement, high levels good for graft survival but poor for cancer
Biomarker analysis by urinary flow cytometry
- Since biopsies are highly invasive in our immunocompromised patients we are trying to use urine (in research only) as a way of monitoring rejection - would be better for patient
What is PTLD
Post-transplant lymphoproliferative disorder
Caused by EBV
How can flow be used for therapeutic monitoring?
FC can determine immunosuprression-dependent inhibition of T cell activation upstreak of cytokine production
- phospho-flow and image flow cytometry (IFC)
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