Flow Cytometry - Introduction and applications Flashcards
What is flow cytometry?
Flow cytometry = A technique used to simultaneously measure several physical characteristics of a single cell in suspension.
Done by light scatter and fluorescence.
What is the definition of flow cytometry?
Measuring properties of cells in flow
What is the definition of flow sorting ?
Sorting (separating ) cells based on properties measured in flow.
aka Fluorescence-Activated Cell Sorting (FACS)
What information can flow cytometer tell us about a cell?
- Relative size
- Relative Granularity/Internal Complexity
- Relative fluorescence intensity (using markers)
What markers can be measured ?
- Cell surface receptors
- Intercellular cytokines
- DNA-Apoptosis/cell cycle/viability
What are two methods of visualisation?
- Flow cytometry
- Fluorescence Microscopy
What technique can we use to quantitate cells?
Flow cytometry - quantifies fluorescence
How does a flow cytometry work?
Fluidics :
-Cell in suspension flow through in a single file.
Optics:
- Cells are hit by laser
- An illuminated volume where they scatter light and emit fluorescence that is collected and filtered.
Electronics:
Light signals are converted into digital values that are stored on a computer
(Then analysed later)
Outline the different processes involved in flow cytometry
Light source Flow chamber Optical system Light detectors Computer
Describe the Fluidics phase
- Cells must be in suspension and flow in single file. Achieved by:
- The sample is injected into sheath fluid through a small orifice (opening)
- Sample fluid flows in a central core that does not mix with the sheath fluid (Laminar flow)
- Introducing a large volume into a small volume (Hydrodynamic Focusing)
slides 12 + 13
Describe the Light source for the Optic stage.
Light source = laser:
- Single wavelength of light (laser) /mix of wavelengths (rare)
- Can provide milliwatts-watts of light
- Inexpensive air-cooled units/Expensive water cooled units
- emit coherent light (single frequency)
Describe light scatter
Light scatter occurs when light hits the cell.
- Most common wavelength of laser is 488nm.
- The light becomes scattered in a forward direction (FSC) and this is proportional to cell size
- Light is also emitted at a 90 degree angle to cell
- 90 degrees light scatter proportional to granularity of cell = SSC Side scatter
no fluorochromes, just laser hitting cell
Describe dot plot
X axis = forward scatter (size)
Y axis = side scatter (granularity )
Each dot is a cell.
We can identify different type of cells based on their size and granularity.
The scatter shows this information - blood - characteristic scatter
What is Granularity ?
Granularity = A cell’s internal complexity
Describe the Passage of Laser-Based Flow Cytometry
1.Flow cell - cells leaving the nozzle tip are hit by the laser
- The cells have been labelled with fluorochromes that emit different-coloured light.
- There is an overlap in emission spectrum of each fluorochrome.
3.Mirrors and filters restrict the amount of light hitting the PMT (photomultiplier tube) = we can differentiate the fluorescence from each fluorochrome separately
see ppt slides 13 + 17
What is the electronic stage of flow-cytometry?
Electronic stage:
- Final stage
- PMT + computer converts light(analogue) signals → digital signals.
What are the key differences between flow cytometry and fluorescence microscopy?
- Fluorescence microscopy (FM) has many fields which means its not quantitative as you would need to look at many fields to quantify cells accurately.
- Flow cytometer (FC) can analyse 1000s of cells every second = more accurate
- FC quantifies rare cells accurately
- FM = difficult by eye (brightness of cells) using FM but FC quantifies the fluorescence of each cell
slide 8
How does fluorescence happen?
What is this process called?
Fluorescence:
-A laser hits fluorochrome, exciting it at a specific wavelength.
-Fluorochrome then returns to its unexcited state, emitting fluorescence at a higher wavelength.
This is called excitation-emission spectrum
What is stokes shift ?
Stoke’s shift = The energy difference between the lowest energy peak of absorbance and the highest energy peak of emission
What are some different fluorochromes and dyes ?
FITC (Fluorescein isothiocyanate)- Green
PE (Phycoerythrin)-Orange
PerCP (Peridinin Chlorophyll Protein)-Red
What are the wavelengths at which each of these fluorochromes emit/are excited?
FITC - Excited at 488nm and emits at 520nm (green)
PE-Excited at 488nm and emits 580nm (orange)
PerCP- Excited at 488nm and emits at 620nm (red)
What is fluorescence ?
Fluorescence = the emission of light by a substance that has absorbed light/other electromagnetic radiation
Why can different fluorochromes be used together and what is a benefit of this ?
- We can use 3 fluorochomes together, all excited by the same laser but they emit at different wavelengths.
- Emission of diff. fluorochromes always overlaps therefore the overlapping light is filtered out by mirrors + filters so we can analyse data from all 3 fluorochromes together
What are some cells which are in single cell suspension ?
Peripheral blood Bone Marrow Fine Needle aspirate CSF (+ other fluids) Cell lines in single suspension Fresh tissue
What are the 2 ways to label cells with monoclonal antibodies/fluorocromes?
Direct method: Fluorochrome is conjugated onto the monoclonal antibody, which binds directly to the antigen on the cell surface
Indirect method: Fluorochrome is conjugated to a secondary antibody, which then binds to the primary monoclonal antibody, which binds to the antigen
see ppt slide 31
How is the data displayed when converted from light to digital ?
1.Histogram - 1 dimensional display (y axis = cell number, x axis = fluorescence intensity). 1 parameter
2.Dot plot - 2 dimensional
(x axis = side scatter, y axis = forward scatter)
2 parameters - We could potentially identify 4 populations - slide 33
What are two things could do to the data we have collected on computer?
Gating
Analysis
What is Gating ?
- 2D dot plot
- Draw a gate around the population we want to analyse.
- Then computer only displays the gated cells by their fluorescence – both FITC + PE
- Identify 3 cell populations
What is Analysis?
Analysis:
- Computer only measures cells satisfying the gate - only analysing one parameter (FITC fluorescence, histogram)
- Use a marker to see what proportion of the cells are negative/positive for it .
Outline how increasing fluorochromes can increase identified populations
1 fluorochrome = 1 population
2 fluorochrome= 4 populations
3 fluorochrome = 8 populations
Cells which are positive for all 3/negative for all 3/all combinations
How can we analyse cell cycles?
→Fluorescent(fluorochrome) dye (PI) increases fluorescence when it binds to cellular DNA.
→Must permeabilise plasma membrane for PI to enter cell
How is propidium iodide used ?
PI is excited by a 488nm laser and emits at 620nm (red)
What are the different stages of the cell cycle?
G0, G1,S,G2,M
What is propidium iodide fluorescence proportional to ?
Propidium iodide fluorescence is proportional to the amount of DNA in a cell.
-G2/M phase has highest fluorescence as cells have double the DNA (compared to G0/G1)
Use PI to quantitate the proportion of cells in different stages of the cell cycle by drawing markers around these 3 pops and asking computer to tell us what proportion of cells are in each stage of cell cycle
How can we measure cell viability ?
- PI cannot cross cell membrane
- If PI penetrates cell membrane = damaged cell.
- PI enters cell and stains DNA
- Cells that are brightly fluorescent with PI are damaged/dead cells
Describe apoptosis briefly
Apoptosis is a programmed cell death where the cell goes through regulated process of dying
Characteristics are condensation of the chromatin material
Blebbing of nuclear material
Accompanied by internucleosomal degradation of DNA which gives rise to ladder pattern on DNA gel electrophoresis
3 methods to detect apoptosis in cells
- Stain cells using PI dye - fix cells for PI to enter
- Phosphatidyl serine (this is usually inside cell membrane but during apoptosis will be on the outside) can be detected by incubating the cells with fluorescien-labeled Annexin V,PI - don’t fix cells
- Stain with 7-aminoactinomycin - don’t fix cells
Describe the PI dye method of detecting apoptosis
Apoptotic cells have sub-G0 peak
-detected using PI stain
Problem: Not all cells have this peak and
The peak may be DNA fragments
Describe the Annexin V + PI method of detecting apoptosis
Live cell = Phosphatidyl serine normally sits inside the cell, intact membrane. - annexin, - PI
During early apoptosis = PS will be outside = Annexin V can bind to it but the cell membrane is intact. + annexin, - PI
During late apoptosis = PS is still on the outside, Cell membrane is no longer intact = PI enters cell. + annexin, + PI
Describe the 7-Aminoactinomycin D method -7-AAD (apoptosis)
- Excited at 488nm
- Emitted at 660nm (red)
- Dye to measure apoptosis
- DNA-specific
- Intercalates in G-C regions
- Long emission wavelength
A single dye - allows us to use 2 other fluorochromes to evaluate cell surface antigens as well as apoptosis
List some applications of flow cytometry.
- Immunophenotyping of leukaemias & lymphomas. Use antibody panels flow cytometry to determine type of leukaemia that the patient has
- Detection of MRD
- Stem cell enumeration
CD4/CD8 in HIV - Measurement of intracellular cytokines
- Study of cell cycle, viability & apoptosis
- Measurement of cell proliferation
- Assessment of transfection efficiency
Describe the fine-tuning of cell sorting
The basics of flow cytometry are present: Cells are in suspension in single file, laser hits cells = cells emit light, which is detected and a computer connected to it determines what type of cell it is.
In cell sorting, we can draw a range around the types of cells we want, so the computer will ‘sort them out’.
The nozzle tip is always vibrating - so much that the stream breaks off into droplets at some point.
Within milliseconds of detecting a certain cell, if it is the type that we want, the computer will charge that cell when it is at the end of a droplet, breaking it off.
The charged droplet cell is then collected into a tube by being pulled towards a deflection plate (due to its charge). The rest of the cells the go to be collected for waste.
= We obtain a sample of very purified cells, with research potential.
Describe the simplest univariate cell cycle method.
→Fluorescent(fluorochrome) dye (PI) increases fluorescence when it binds to cellular DNA.
→Must permeabilise plasma membrane for PI to enter cell
