Flow Cytometry: Introduction And Applications

Question 1

Define flow cytometry

Answer 1

• A Technique which simultaneously measures several physical characteristics belonging to a SINGLE CELL in SUSPENSION in flow.
• Done via light scattering and fluorescence

Question 2

Define flow sorting

Answer 2

• Sorting (separating) cells based on properties measured in flow
• Also called Fluorescence-Activated Cell Sorting (FACS)

Question 3

Identify the key differences between flow cytometry and fluorescence microscopy?

Answer 3

• Flow cytometry essentially allows for quantification of different metrics compared to fluorescence microscopy e.g.
• Quantifies number of cells
• Identification of rare cells
• Quantifies fluorescence with a measurement

Question 4

Basics of flow cytometry
Outline what flow cytometry can tell us about a cell?

Answer 4

• 1. Its Relative Size
• 2. Its Relative Granularity/Internal Complexity
• In terms of organelles present in cell
• 3. Its Relative Fluorescence Intensity
• How much light a cell can emit?

Question 5

Outline the major components of flow cytometry instrumentation?

Answer 5

• Fluidics: Cells in suspension flow in single-file through
• Optics: an illuminated volume where they scatter light and emit fluorescence (via stained cells) that is collected and filtered
• Electronics: converted to digital values that are stored on a computer

Question 6

Describe the fluidics component of flow cytometry?

Answer 6

• Need to have cells in suspension flow in single file
• Accomplished by injecting sample into a sheath fluid as it passes through a small (50-300 um) orifice
• Sample fluid flows in a central core that does not mix with the sheath fluid - Laminar flow
• Introduction of a large volume into a small volume - Hydrodynamic Focusing
  HF = faster moving sheath fluid (saline solution) forces sample into smaller core strea, where all particles travel along the same axis at the same velocity

Question 7

Describe the laser light source of optics in flow cytometry?

Answer 7

• Single wavelength of light (a laser line) or (more rarely) a mixture of wavelengths
• Can provide from milliwatts to watts of light
• Can be inexpensive, air-cooled units or expensive, water-cooled units
• Provide coherent light (Single frequency)

Question 8

Describe the different scattering pattern of light in flow cytometry and what this shows?

Answer 8

• Increase Forward scatter = Increase size of the cell
• Increase size scatter = increase complexity/granularity of cell

Question 9

Describe the electronic in flow cytometry?

Answer 9

Processing of light signals from detectors via analog-digital conversion (ADC) converter)

Question 10

Define what fluorescence is.

Answer 10

• Also known as stokes shift
• Energy difference between the lowest energy peak of absorbance and highest energy of emission
• Emission is the release of light (scattering) when excited at high levels after absorance occurs

Question 11

List some commonly used fluorochromes

Answer 11

• Fluorescein isothiocyanate (FITC) - GREEN
• Phycoerythrin (PE) - ORANGE
• Peridinin Chlorophyll Protein (PerCP) - RED

Question 12

What is special about Fluorochromes?

Answer 12

Fluorochromes used in flow cytometry are essentially those that can attach in some way to biologically significant molecules and are excitable by the lasers commonly found on commercial flow cytometers which emit a specific light

Question 13

Demonstrate an understanding of fluorescence excitation and emission
Demonstrate knowledge of the excitation and emission spectra between PE, FITC, PRCIP VD

Answer 13

• The excitation spectrum shows at what wavelengths the solution is used to produce its fluorescence.
• The emission spectrum shows what wavelengths are given off from the solution

Question 14

Explain why combinations of fluorochromes can be used together, and the benefits of such use of combinations of fluorochromes.

Answer 14

• 3 FLC all emit light at different wavelengths
• Allows for filtering of overlapping light so analysis of 3 flurochromes can occur for the cell

Question 15

List common sources of cells for flow cytometry (5)?

Answer 15

SINGLE CELLS IN SUSPENSION
- Peripheral blood
- Bone marrow
- Fine Needle Aspirate
- CSF and other fluids
- Fresh Tissue

Question 16

Describe and differentiate between direct and indirect fluorescence labelling.

Answer 16

• DIRECT: Monoclonal antibodies (Mobs) are preconjugated to fluorochromes
• INDIRECT: Unconjugated MoAbs
• Involves primary and secondary antibody
• Primary linked to secondary which conjuagtes with fluorochrome

Question 17

What two ways can data be displayed in flow cytometry, describe their graph?

Answer 17

• Histogram (1D) or dot plot (2D)
• Dimensional as in how many parameteres it measures
• Histogram - X= cell number, Y= fluorescence intensity
• Dot plot - X= Side scatter Y= Forward scatter

Question 18

Which graph type is better?

Answer 18

Dot plot > H as it measures 2 different paramteres at once, whereas H only does one (fluorescence intensity)

Question 19

State the two methods that occur when dot plot has been produced in flow cytometry?

Answer 19

• Gating
• Analysis

Question 20

State the gating method that occur when dot plot has been produced in flow cytometry?

Answer 20

• Gate a specific area of cells in a shape
• Gate cells in a graph of 2 fluorochromes where it allows for identification of number of populations

Question 21

State the analysis method that occur when dot plot has been produced in flow cytometry?

Answer 21

• 1) Analyse one of those populations in a graph of one of the flurochromes - shows proportion of cells which are positives or negatives for parameter
• Bar on graph indicates how much of the cells contain or satisfy the fluorochrome
• 2) Split gated graph into 4 quadrants where each area indicates -ve or + ve populations for the specific FCs

Question 22

Describe what each quadrant of a gated analysis graph indicates

Answer 22

• Single positive PE population: Top left
• Negative population: Bottom left
• Double positive FITC and PE population: Top right
• Single positive FITC population: Bottom right

Question 23

How many populations can be identified if 3 antibodies are used for the cells?

Answer 23

8

Question 24

Describe cell cycle analysis using flow cytometry, and some commonly used fluorochromes and how its fluorescence varies throughout the cell cycle?

Answer 24

• Cellular DNA is detected using a fluorescent dye that binds preferentially to DNA
• Propidium iodide is most commonly used. It undergoes a dramatic increase in fluorescence upon binding DNA. It requires permeabilization of the plasma membrane.
• To allow for entry into the membrane
• Its fluorescence increases dependent on DNA - remember limited amount of propidium iodide

Question 25

Describe how flow cytometry can be used for assessment of cell viability and the fluorochrome used for this?

Answer 25

- Propidium iodide (PI) cannot normally cross the cell membrane - 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

Question 26

Describe how flow cytometry can be used for the quantitation of apoptosis, and some commonly used fluorochromes.

Answer 26

- 3 key methods: - By staining with the dye PI (cells fixed) - Phosphatidyl serine, can be detected by incubating the cells with fluorescein-labeled Annexin V, and PI (cells not fixed) - PS is released from within the cell when the cell apoptoses - By staining with 7-aminoactinomycin D (cells not fixed)

Question 27

Describe the propidium iodide method for apoptosis and why it isn't a reliable method?

Answer 27

- Here there is a peak at sub G zero which indicates apoptopic cells (VD) - Not reliable method - Could be DNA fragments - Some cells don't display sub G0 peak

Question 28

Describe the detection via Annexin V and PI system for apoptosis comparing against live, early apoptoic and late apoptoic

Answer 28

VD

Question 29

Describe the 7-AAD method

Answer 29

- 7-AAD = 7-Aminoactinomycin D (7-AAD) - Ex: ~488 nm - Em: ~660 nm - DNA-specific - intercalates in G-C regions - Long emission wavelength

Question 30

Why is the 7 AAD method used?

Answer 30

- Single dye with FITC & PE labeled Antibodies - Used for simultaneous evaluation of DNA content and 2-color immunofluorescence (apoptosis measurement) using only 488 nm Ex

Question 31

Describe the machinery of flow sorting

Answer 31

- Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry. It provides a method for physically separating defined cell populations without cultivation - It sorts a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell from the analyser

Question 32

What are the applications for the machinery of flow sorting?

Answer 32

- Cell sorting is used for a wide range of applications, from profiling immune subsets to sorting cancer cells and screening CRISPR libraries, to purifying engineered microbes and enriching nuclei for single cell epigenomic

Question 33

State the clinical applications of flow cytometry

Answer 33

- Immunophenotyping of leukaemias & lymphomas - Detection of Minimal residual disease - Stem cell enumeration - CD4/CD8 detection in HIV

Question 34

State the research applications of flow cytometry

Answer 34

- Measurement of intracellular cytokines - Study of cell cycle, viability & apoptosis - Measurement of cell proliferation - Assessment of transfection efficiency

Question 35

What is transfection?

Answer 35

Transfection = process of introducing nucleic acids into eukaryotic cells by nonviral methods