Flow Cytometry - Introduction

Question 1

What is Flow Cytometry?

Answer 1

Technique which simultaneously measures several physical characteristics belonging to
a SINGLE CELL in SUSPENSION

This is done by LIGHT SCATTER and 
  FLUORESCENCE   

Measuring properties of cells in flow

Question 2

Flow Sorting - define

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

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

Question 4

Visualisation methods - list

Answer 4

Fluorescence Microsocopy

Flow Cytometry

Question 5

Basics of Flow Cytometry - brief process

Answer 5

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

Question 6

Describe the flow of cells in suspension and its control

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

Question 7

Laminar flow - define

Answer 7

Sample fluid flows in a central core that does not mix with the sheath fluid - Laminar flow

Question 8

Hydrodynamic Focusing - define

Answer 8

Introduction of a large volume into a small volume - Hydrodynamic Focusing

Question 9

Describe what is detected after laser light source applied

Answer 9

Fluorescence emitted from stained cells detected

Forward and side scattered light from all cells detected

Question 10

Describe characteristics and +ves of lasers

Answer 10

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 11

Stokes Shift - define

Answer 11

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

Question 12

FLUOROCHROMES and DYES - list them

Answer 12

Fluorescein isothiocyanate (FITC) GREEN

Phycoerythrin (PE)
ORANGE

Peridinin Chlorophyll Protein
RED (PerCP)

Question 13

Describe composition of single cell suspension

Answer 13

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

Question 14

Compare direct/indirect labelling

Answer 14

DIRECT : Monoclonal antibodies (MoAbs) are preconjugated to fluorochromes

INDIRECT: Unconjugated MoAbs (bound to secondary antibody which is bound to flurophore)