L8: Flow Cytometry Flashcards by BLACK H

What is flow cytometry?

→Measuring properties of cells in flow

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What is flow sorting?

→Sorting (separating) cells based on properties measured in flow

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What is another name for flow sorting?

→Fluorescence-Activated Cell Sorting (FACS)

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What can a flow cytometry tell us about a cell?

→Its Relative Size
→Its Relative Granularity/Internal Complexity
→Its Relative Fluorescence Intensity

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What proteins can be used to identify specific cell subsets?

→cytokine or chemokine
→metabolic proteins
→adhesion molecules

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What are the methods of visualisation?

→Fluorescence Microscopy

→Flow Cytometry

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What are the differences between the methods of visualisation?

→Microscopy- have to scan many fields but FC can analyse thousands of cells.

→FC can detect rare cells.

→FC quantitates the amount of fluorescence in each cell

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What can the basics of flow cytometry be divided into?

→fluidics
→optics
→electronics

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What are the basics of FC?

→Cells in suspension
flow in single-file 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

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What are the basic stems of FC?

light source→ flow chamber→ optical system→ light detectors→ computer

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How are cells suspended in a single file?

→by injecting sample into a sheath fluid as it passes through a small (50-300 µm) orifice
→hydrodynamic focusing

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What is hydrodynamic focusing?

→Introduction of a large volume into a small volume
→when two streams of fluids with different flow rates are running side-by-side and in the same direction into a flow cell, then a laminar flow is created

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What is laminar flow?

→Sample fluid flows in a central core that does not mix with the sheath fluid

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What is involved in the optics stage of FC?

→lasers

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Describe lasering of the cells

→Single wavelength of light (a laser line) or (more rarely) a mixture of wavelengths
→can provide from milliwatts to watts of light

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What sort of frequency is the wavelength of light in the lasers?

→Single frequency

→coherent light

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Which is more expensive? Air cooled units or water-cooled units?

→water-cooled units

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What is the meaning of the forward light scatter?

→proportional to size

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What is the meaning of the side scatter?

→granularity

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What are dichroic filters?

→direct certain wavelengths of light one way, while deflecting other wavelengths

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What are PMTs?

→amplifies the signal

→photomultiplier tubes

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What is involved in the electronics part of FC?

→Processing of signals from detectors

→Analog-Digital Conversion

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What is Stokes shift?

→the energy difference between the lowest energy peak of absorbance and the highest energy of emission

What is fluorescence?

→absorb light at a particular wavelength and then emit it at a higher wavelength.

What are the different fluorochromes and their dyes?

→Fluorescein isothiocyanate (FITC)= GREEN →Phycoerythrin (PE)= ORANGE →Peridinin Chlorophyll Protein (PerCP) =RED

What filters out the overlap between the fluorochromes?

→dichroic mirrors

Which dye has the highest emission wavelength?

→PerCP

Which cells are in single cell suspension?

``` →Peripheral blood →Bone marrow →Fine Needle Aspirate →CSF and other fluids →Fresh Tissue ```

What are two types of labelling in immunofluorescence?

→direct | →indirect

What is direct labelling?

→Monoclonal antibodies (MoAbs) are preconjugated to fluorochromes

What is indirect labelling?

→Unconjugated MoAbs →uses two antibodies. →The primary antibody is unconjugated and a fluorophore-conjugated secondary antibody directed against the primary antibody is used for detection.

What kind of dimension is a histogram?

→one dimensional display →cell number on x-axis, intensity on y-axis →Only interrogate one fluorescence at a time

What is gating in FC analysis?

→draw a gate around population of interest and the computer displays only cells in that gate

What stain is specific for neutrophils?

→CD59 antibody

How many populations can be resolved by 3 antibodies?

→8 | →More antibodies, more flurochromes, more populations can be detected

What is propidium iodide?

→It undergoes a dramatic increase in fluorescence upon binding DNA

What does propidium iodide require for binding?

→requires permeabilization of the plasma membrane- punch holes for propidium to get into cells

At what wavelength does PI excite and emit?

→excite at 500nm | →emit at 600nm

How are the number of cells and their content in the cell cycle quantitated?

→stained with PI

What goes on the x and y-axis of cell cycle quantitation graphs?

→X-axis= intensity of fluorescence- proportional to amount of DNA in a cell →Y-axis= cell count

How does PI assay work?

→If the PI penetrates the cell membrane, it is assumed to be damaged →Cells that are brightly fluorescent with the PI are damaged or dead

What are the characteristics of apoptosis?

→condensation of the chromatin material →Blebbing of nuclear material →internucleosomal degradation of DNA →'ladder' pattern on DNA gel electrophoresis

What are the three ways apoptosis is detected?

→staining with the dye PI (cells fixed) →incubating the cells with fluorescein-labeled Annexin V, and PI →By staining with 7-aminoactinomycin D

Why do apoptosis have ladder appearance on gel electrophoresis?

→internucleosomal degradation of DNA

Why is phosphatidyl serine(PS) used to detect apoptosis?

→normally found on the inside of the membrane | →when cells are apoptosis, it is expressed on the outside.

How is PS used to detect apoptosising cells?

→incubating the cells with fluorescein-labelled Annexin V, and PI →cells not fixed

What is the difference between using PI staining and annexin V incubation and staining by 7AD?

→PI requires cells to be fixed → annexin doesn't require fixing →7AD- cells not fixed

What peak do apoptotic cells display on PI graphs?

→sub-50 peak

Why are sub-G0 peaks not reliable in detecting apoptotic cells?

→peak can be DNA fragmentation. | →Not all apoptotic cells show that peak

What are the excite an emission wavelengths of 7AAD?

→Ex: ~488 nm | Em: ~660 nm

Where does 7AAD intercalate?

→ intercalates in G-C regions

Which fluorochromes can be used with 7AAD?

with FITC & PE labeled Ab for simultaneous evaluation of DNA content and 2-color immunofluorescence using only 488 nm Ex

Are live cells negative or positive for 7ADD?

→negative

What happens to the nozzle tip in cell sorting and why?

vibrated so cells break off into droplets containing single cells

What happens when the cell of interest gets to the last drop?

→charge is applied and pulled by deflection plates into a specific tube

What increases after sorting in FC?

→Purity has increased after sort | →Allows purification of rare cells

What are the applications of flow cytometry?

``` Immunophenotyping of leukaemias & lymphomas →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 ```