Flow Cytometry

Question 1

What is Flow Cytometry?

Answer 1

Technique which simultaneously measures several physical characteristics belonging to a SINGLE CELL in SUSPENSION
– Measuring properties of cells in flow

This is done by LIGHT SCATTER and FLUORESCENCE

Question 2

Flow Sorting

Answer 2

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

Question 3

WHAT CAN A FLOW CYTOMETER TELL US ABOUT A CELL?

Answer 3

1. Its Relative Size
2. Its Relative Granularity/Internal Complexity
3. Its Relative Fluorescence Intensity

Question 4

METHODS OF VISUALISATION

Answer 4

• Fluorescence Microsocopy

- Flow Cytometry

Question 5

Basics of flow cytometry

Answer 5

Fluidics
•Cells in suspension
•flow in single-file through

Optics
•an illuminated volume where they
•scatter light and emit fluorescence
•that is collected, filtered and

Electronics
•converted to digital values
•that are stored on a computer

Question 6

Fluidics

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 μm) 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

Question 7

Optics - Light Sources

Answer 7

Lasers
– 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)
diagrams

Question 8

Electronics

Answer 8

Processing of signals from detectors

– Analog-Digital Conversion

Question 9

Stokes Shift

Answer 9

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

Question 10

IMMUNOFLUORESCENCE

Answer 10

FLUOROCHROMES and DYES

Fluorescein isothiocyanate (FITC) = GREEN

Phycoerythrin (PE) = ORANGE

Peridinin Chlorophyll Protein (PerCP)= RED

Question 11

SINGLE CELLS IN SUSPENSION

Answer 11

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

Question 12

METHOD OF LABELLING

Answer 12

DIRECT : Monoclonal antibodies (MoAbs) are preconjugated to fluorochromes
INDIRECT: Unconjugated MoAbs
diagrams

Question 13

Univariate Cell Cycle Methods

Answer 13

• In the simplest method, 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.

Question 14

Propidium Iodide (PI)

Answer 14

How the assay works: Cell Viability
• 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 15

Measurement of Apoptosis

Answer 15

• Apoptosis is programmed cell death where the cell goes through a highly regulated process of “dying”
• Characteristics are condensation of the chromatin material
• Blebbing of nuclear material
• Often accompanied by internucleosomal
degradation of DNA giving rise to distinctive ‘ladder’ pattern on DNA gel electrophoresis

Question 16

Detection Methods for Apoptosis

Answer 16

• 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)
• By staining with 7-aminoactinomycin D (cells not fixed)

Question 17

7-Aminoactinomycin D (7-AAD)

Answer 17

• Ex: ~488 nm
• Em: ~660 nm
• DNA-specific
–  intercalates in G-C regions
• long emission wavelength 
– with FITC & PE labeled Ab for simultaneous evaluation of DNA content and 2-color immunofluorescence using only 488 nm Ex

Question 18

Applications

Answer 18

• 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