Cell based methods including flow cytometry Flashcards
Common methods in Cellular Immunology
- Isolation of cells
density gradient centrifugation, magnetic beads, flow cytometry - Viability/apoptosis
- Microscopy
cell counting, immunofluorescence and immunohistochemistry - Characterisation of (lymphocyte) specificity and function
proliferation, ELISPOT, Luminex, flow cytometry
buffy coat
The buffy coat (1% of total blood)is the fraction of an anticoagulated blood sample after centrifugation that contains most of the white blood cells and platelets
Leukocyte types and estimated frequency
-CD4+ T cells (CD3, CD4)
- CD8+ T cells (CD3, CD8)
–>40-70%
B cells (CD19, Ig+) –> 5-10%
NK cells (CD56) <5%
Monocytes, macrophages (CD14) –>5-20%
Dendritic cells subtypes < 1%
Basophils < 1%
name 2 ways to Isolate cells with magnetic beads
Direct labeling - fastest way of magnetic labeling, one labeling step since the specific antibody is directly coupled to the magnetic particle.
Indirect labeling - if no direct microbead for the cell type of interest is available or if different cells should be depleted from the sample.
what is Positive isolation in Isolation of cells with magnetic beads
Positive isolation: label the target cell
pros:
- reduce the number of washing steps and avoids unnecessary cell loss
- maximum purity
- ideal for flow analysis or molecular applications
cons:
- may activate the targeted cells
- the marker used can be saturated and not available for later stimuli
what is Untouched/Negative isolation in Isolation of cells with magnetic beads
Untouched/Negative isolation: label the unwanted cells
pros:
- truly untouched cells, bead- and antibody-free
- high viability
- easy removal of unwanted cell types
cons:
difficult to get high yield and purity for small target populations
MACS cell
separation technology
can either use positive or negative isolation to isolate the desired cells from the sample.
Small: 50 nm
Biodegradable: composed of a
biodegradable matrix; no need to remove
them after the separation process
Straight to experiment: have no known
effect on structure, function, or activity
status of labeled cells and do not interfere
with subsequent experiments
Dynabeads cell separation technology
Large: 1 μm to 4.5 μm
preferred application is negative cell selection, where the cells targeted by the
magnetic label are discarded, and only the unlabeled cells are used, or positive cell
selection if the magnetic bead is detached.
cell viability
living cells
cell counting cells by trypan blue, how do you determine the cell viability?
Cell concentration (cells/ml) = V x D x 10^4
V: trypan blue negative cells
D: dilution factor
T: total number of cells
Cell viability (%)= V/ T x 100
Dead cells. Trypan blue positive
Viable cells. Trypan blue negative
Immunohistochemistry - IHC
Immunohistochemistry is the localization of proteins/antigens in tissue sections by the use of labeled antibody as specific reagents through antigen-antibody
interactions.
The antibody is chemically attached to an enzyme that converts a colorless
substrate into a colored reaction product in situ. The colored product can be
visualized with a light microscope.
Most commonly used enzymes are horseradish peroxidase (brown) and alkaline phosphatase (red or blue)
Immunofluorescence microscopy
visualize the location of proteins in tissue sections by the use of antibodies conjugated to fluorescent dyes.
can use Direct or indirect labeling
Confocal microscopy
computer aided imaging technique to
produce ultra-thin optical section of a cell or
tissue.
Three-dimensional image of the sample can
be obtained.
Time-lapse video microscopy
sensitive digital cameras record the movement of fluorescently labeled molecules in cell membranes and their distribution when cells come into contact with each other.
how many markers are used in standard IHC/IF?
Traditionally 2-3 markers are used
high-multiplex tissue imaging
Different technologies available:
Fluorophore tagged: 100+ markers
DNA tagged: 50-100 markers
Metal isotope tagged: 40-50 markers
3H-thymidine incorporation assay
To characterize lymphocyte specificity and function, particularly focusing on proliferation.
Spin cells 1200 rpm 5 min
Count and seed cells
1. Add the drug (some left
unstimulated as control)
2. Incubate cells
3. Pulse them with tritiated
3H-thymidine
3H-thymidine is incorporated into DNA when cells divide -> high level of cell division
(activated T-cells that divide) leads to high level of radioactivity
- Harvest the cells
- Evaluate radioactivity in a scintillation counter (higher level = higher proliferation)
Cell trace staining
Cell trace staining is a method used to monitor cell proliferation by labeling cells with a fluorescent dye that can be tracked through several generations of cell division.
Cell trace staining steps
Spin cells 1200 rpm 5 min
Stain with cell trace
Count and seed cells
- Add the drug (some left
unstimulated as control) - Incubate cells
- Analyse in flow
cytometry
ELISPOT assay
Simple tool to measure frequency of T cell responses
to detect and quantify individual cells that secrete a particular cytokine or other molecules. useful for measuring the frequency of cytokine-secreting cells in a population, such as T cells
ELISPOT can also be used to detect specific antibody secretion by B cells by using antigen- coated surfaces to trap specific antibody (Ab) and labeled anti-Ab
explain the steps in ELISPOT assay
1.Cytokine specific specific antibodies are bound to the surface of a plastic well
2.Activated T cells are added to the well. these T cells are a mixture of different effector functions.
- Cytokine secreted by some activated T cells is captured by the bound antibody.
- The captured cytokine is revealed by a second cytokine specific antibody, which is coupled to an enzyme, giving rise to a spot of insoluble coloured precipitate.
Counting the number of spots and knowing the number of T cells originally added to the
plate allows a simple calculation of the frequency of T-cells secreting the particular cytokine
Cytometry
refers to the measurement of physical and/or chemical characteristics of cells or other biological particles
Characteristics, examples:
Cell size, shape and internal complexity
Cell markers (CD3, CD4, CD14, CD19)
Cytokine production (IL-4)
Cell cycle (DNA content)
Apoptosis/viability (Membrane integrity)
Activation (calcium flux
Flow Cytometry
is a process in which such measurements are made while the cells or
particles pass through the measuring apparatus in a fluid stream
- Cells need to be in suspension.
- Thousands of cells can be analysed per second
Flow Sorting
extends flow cytometry by using electrical or mechanical means to divert and
collect cells with one or more measured characteristics falling within a range of values
Sorting: When a cell meeting the specified criteria is identified, the system uses one of the following sorting mechanisms to direct the cell into a collection container:
Electrostatic Deflection: The flow cytometer applies a charge to droplets containing cells as they break off from the stream. Deflection plates generate an electric field that diverts charged droplets into designated collection tubes based on their charge.
Mechanical Sorting: The system uses mechanical gates or channels to physically separate cells.
name some applications of The Flow Cytometer
Phenotype of cells, surface molecules, intracellular cytokine staining, cell signaling molecules, cell sorting, cellular pH, calcium flux assays, apoptosis analysis, cell cycle analysis, cell proliferation, cellular transport assays, drug
uptake/efflux assays, transfection efficiencies, phagocytosis assays, etc.
What is in a flow cytometer?
Fluidics =To focus the cells
for interrogation by a laser
Optics = To generate and collect the light signals (scatter and fluorescence)
Electronics = To convert the optical signals to electronic
signals and digitize them
for computer analysis
(PMTs)
What can a flow cytometer tell us about a cell?
-Its relative size
(Forward Scatter—FSC)
-Its relative granularity or internal complexity
(Side Scatter - SSC)
How is a dot plot generated?
A dot plot in the context of flow cytometry is a graphical representation that shows the relationship between two parameters (e.g., forward scatter and side scatter, or two different fluorescence channels) for each cell analyzed. Each dot on the plot represents a single cell, allowing researchers to visualize and interpret complex data from heterogeneous cell populations.
x- axis is forward scatter (represents size)
y-axis is side scatter(represents granularity)
Double-positive cells (expressing both markers) will have high values on both axes and will appear in the top-right quadrant.
Double-negative cells (expressing neither marker) will have low values on both axes and will appear in the bottom-left quadrant
Fluorochromes
components of molecules which absorb light and emit fluorescence
They typically contain aromatic groups
how does fluorescence occur?
Absorption of a photon raises the electron from ground state to an excited state.
Emission of photons as the electron returns from an excited state to ground state
gives rise to the fluorescence.
what can Antibodies labeled with fluorochromes be used for?
used for marking cell surface and intracellular antigens
(Common labels: FITC, PE, APC)
Tandem dyes
two covalently attached fluorescent molecules (donor and acceptor) e.g. Pe/Cy7
* behave as a unique fluorophore - excitation properties of donor, emission
properties of acceptor
what does the Analysis of flow cytometry look like?
- PMT (photomultiplier tubes) signals are converted to digital information and
processed in a computer. - The information can be viewed as histograms, dotplots, density plots or
contour plots.
How is a dotplot generated? Fluorescence PE vs FITC (two markers used)
Negative Population in the bottom left square
Single Positive PE Population in the top left square
Double Positive Population in the top right square
Single Positive FITC Population in the bottom right square
How is a histogram generated?
Fluorescence from
one channel, i.e. FITC
Viability/apoptosis
Annexin-V staining: early apoptosis
Annexin-V staining is a commonly used method to detect early apoptosis in cells. Annexin-V is a protein that binds specifically to phosphatidylserine (PS), a phospholipid that is normally found on the inner leaflet of the plasma membrane. During apoptosis, PS is translocated from the inner to the outer leaflet of the plasma membrane, exposing it to the external environment. Annexin-V can bind to PS with high affinity, making it a useful marker for detecting apoptotic cells
7-amino-actinomycin D (7-AAD) and propidium iodide (PI)
vital dyes that bind to nucleic acids, such as DNA, in cells. They are commonly used in flow cytometry and fluorescence microscopy to distinguish between viable, apoptotic, and necrotic cells based on membrane integrity.
These dyes can only penetrate the plasma membrane when the membrane integrity is compromised, such as in the later stages of apoptosis or during necrosis. In viable cells with intact membranes, these dyes are unable to enter the cells.
- Bind to nucleic acids
- Can only penetrate the plasma membrane when membrane integrity is
breached, as occurs in the later stages of apoptosis or in necrosis.
What is the viability status of marked cellpopulations?
Live Cells: Cells that are negative for dead cell markers and exhibit normal metabolic activity are considered viable.
Dead Cells: Cells that stain positive for dead cell markers or show decreased metabolic activity are considered non-viable
No Apoptosis (Viable cells)
Cells that are
negative for both Annexin V and 7AAD/PI have no
indications of apoptosis. PS translocation has not
occurred and the plasma membrane is still intact.
Early Apoptosis
Cells that are Annexin V-positive and 7AAD/PI – negative. PS translocation has
occurred, but the plasma membrane is still intact
Late Apoptosis or Cell Death/Necrosis
Cells that are positive for both Annexin V and the 7AAD/PI are either in the late stages of apoptosis or are already dead. PS translocation has occurred and the plasma membrane integrity is lost.
why is Compensation required when using multiple fluorophores whose emission signals overlap?
it helps to correct for spectral overlap and ensure accurate interpretation of fluorescence signals in flow cytometry
Multiplexing in flow cytometry
Multiplex analysis of cells in suspension.
The number of lasers in modern instruments are increasing.
Advanced conventional instruments: 5 lasers -> 21-28 parameters to be measured in parallel.
Fluorochromes continously being developed which enables increased complexity.
You can analyse with gates and subgates, giving detailed information on a sample.
Excitation – what lasers you
have will affect your choice.
Emission spectras have to be
considered when multiplexing. Should overlap as little as possible
Next level for multiplex flow cytometry: SPECTRAL
(instead of conventional described before)
The different fluorochromes have unique spectra. However, the leak of the red spectra into the filter that is supposed to measure the yellow spectra is large -> problem in conventional flow cytometry (Compensation will not be
enough!)
Spectral provides spectral unmixing! Higher multiplexing
possible with spectral!
what are the Important factors to consider in flow cytometry?
These factors are indeed critical considerations in flow cytometry experiments:
Compensation: Correcting for spectral overlap ensures accurate interpretation of fluorescence signals when using multiple fluorophores.
Unspecific Staining: Differentiating between specific and nonspecific binding is essential for obtaining reliable results. Titration of antibody concentrations and appropriate blocking can help minimize nonspecific staining.
Isotype Controls: Isotype controls are crucial for distinguishing specific antibody binding from background signal. They involve using antibodies of the same isotype as the primary antibody but with irrelevant specificity.
Autofluorescence: Cell fluoroscence even when no fluorescent tag or stain is added. Autofluorence comes from normal cell components, such as riboflavin and
flavoproteins
FACS Cell Sorting
- Allows separation of a cells of
interest from a hetero-geneous
population. - Charges are applied to cells of
interest. - Deflection plates attract cells with
opposite polarity
Intracellular cytokine analysis by flow cytometry
– allow the cytokine profile of individual cells to be determined
- Activated T cells are treated with an inhibitor which blocks protein export allowing cytokines to accumulate in the ER
- the cell in fixed and permeabilized with mild detergents.
- Cytokine- specific antibodies penetrate the cell to bind the intracellular cytikines.
Assessment of proliferation using BrdU and intracellular staining
- Synthetic nucleoside that is an
analog of thymidine. - Incorporated into the newly
synthesized DNA of replicating
cells - Can be measured by antibodies towards BrdU (intracellular
staining) - > Frequency of proliferating
cells
(As an alternative to measuring Thymidine incorporation
– but here we need a flow cytometer)
Analysis of secreted cytokines by Cytokine Bead Array (CBA)
Beads with emission wavelength of 650 nm.
Different amounts –> different levels of signal
-> bead (and thus analyte) identification
Labeled antibodies that specifically
capture a particular molecule
The captured analyte is detected by PEconjugated antibodies (max ca 575 nm)’
+ Small sample sizes
+ Very sensitive
+ Short assay time
+ Easy to use
+ Multiplex
+ Does not require special
instrument (if FACS available)
-Expensive
Cytokine analysis by Luminex Multiplex assay
1.Add sample to specific antibody-bead
(serum/plasma or cell supernatant)
2.wash
3. Add biotinylated detector antibody
4. wash
5. Add Streptavidin-PE
6. wash
7. Read in Luminex machine
Total time 3.5 hrs
+ Small sample sizes
+ Very sensitive
+ Short assay time
+ Easy to use
+ Multiplex , >100 analytes
- Expensive
- Requires special
instrument
Identification of TCR specificity using MHC:peptide tetramers
Detect and quantify T-cells that are specific for a given antigen
– cytomegalovirus peptide MHC class I (HLA) molecule
They bind directly to T cell
receptors of a particular
specificity, determined by the
MHC allele and peptide
combination.
MHC class I tetramers –
-for studies of CD8+ T cell responses
MHC class II tetramers –
- for studies of CD4+ T cell responses
