Flow Cytometry - Introduction

Question 1

What is Flow Cytometry?

Answer 1

“Measuring properties of cells in flow:

• Technique which simultaneously measures several physical characteristics belonging to a single cell in suspension.
• This is done by Light Scatter and Fluorescence.”

Question 2

What is Flow Sorting?

Answer 2

“Separating Cells based on properties measured in flow.

Also called Fluorescence - Activated Cell Sorting (FACS).”

Question 3

Which features of a Cell does Flow Cytometer show?

Answer 3

“1. Its relative Size.

2. Its relative Granularity/Internal Complexity.
3. Its relative Fluorescence Intensity.”

Question 4

What can be measured using a Flow Cytometer?

Answer 4

“Cytokines
Enzymes
Adhesion Molecules
Surface Receptors”

Question 5

List the three main components of the Flow Cytometer?

Answer 5

“1. Fluidics:
Cells in suspension flow through in a single-file .

2. Optics:
   An illuminated volume where they scatter light and emit fluorescence.
3. Electronics:
   Collected, filtered and converted into digital values that are stored on a computer.”

Question 6

What is required of the Cells in Fluidics and how is this done?

Answer 6

“1. Need to have cells in suspension.

2. They also need to flow in a single file.
3. Accomplished by injecting a sample into a sheath fluid,
4. As it passes through a small (50-300um) orifice.”

Question 7

Which two Principles does Fluidics use?

Answer 7

“Laminar Flow: Sample fluid flows in a central core that does not mix with the sheath fluid.
Hydrodynamic Focusing: Introduction of a large volume into a small volume.”

Question 8

Describe the Light Source used in Optics:

Answer 8

“1. Single Wavelength of light: a laser line.
or a Mixture of Wavelengths.

2. Can provide from Milliwatts,
   To Watts of light.
3. Can be inexpensive, air cooled units
   Or expensive water cooled units.
4. Must provide coherent light (single frequency).”

Question 9

What is Coherent Light?

Answer 9

“Beam of Photons that all have the same frequency.
Only a beam of laser light will not spread and diffuse.
In lasers, waves are identical and in phase, which produces a beam of coherent light.”

Question 10

How is the Light Scattered in Flow Cytometry?

Answer 10

“Light that is scattered Forward is proportional to the Size of the Cell.

Light that is scattered at a 90 Degree Angle is proportional to the Granularity of the Cell.”

Question 11

How is the result from Optics shown on a Graph?

Answer 11

“The X Axis shows Forward Scatter: Size of Cells.
The Y Axis shows Side Scatter: Ganularity.

Each dot represents an event.
Without any staining you can see certain populations accumulating.”

Question 12

Channel Layout in a Flow Cytometer:

Answer 12

“The Cells have been labelled 4 Colors.
Each color is detected by a different detector.
The emitted light is going through filters and mirror,
So that by the time it gets to the detector it is only detecting a narrow range of wavelengths.”

Question 13

What is Electronics?

Answer 13

“Analog-Digital Conversion:

- Processing of signals from detectors.”

Question 14

What is the Stokes Shift?

Answer 14

The energy difference between the Lowest Energy Peak of absorbance and the Highest Energy of Emission.

Question 15

How is an Excitation Emission Spectrum Produced?

Answer 15

“1. When a Fluorochrome is Excited,
It produces an Excitation Wavelength.

2. When the Fluorochrome returns to its Unexcited state it emits light at a longer wavelength
   This is the Emission Wavelength.

These two Wavelengths produce an Excitation Emission Spectrum.”

Question 16

What is Immunofluorscence?

Answer 16

Method that relies on the use of antibodies chemically labeled with fluorescent dyes to visualize molecules under a light microscope.

Question 17

Which dyes are used in Immunofluroscence?

Answer 17

“Fluorescein Isothiocyanate (FITC) Green
Phycoerythrin (PE) Orange
Peridinin Chlorophyll Protein (PerCP) Red”

Question 18

What are the two methods of labelling in Immunofluroscence?

Answer 18

“1. Direct:
Monoclonal Antibodies are Pre Conjugated to Fluorochromes.

2. Indirect:
   Unconjugated Monoclonal Antibodies.
   You get a lot more background staining with the indirect method.”

Question 19

Compare using a Histogram againsts a Dot Plot?

Answer 19

“1. With a Histogram you can only measure one parameter at a time.
Cell Count is on the Y Axis and Fluorescence is on the X Axis.

2. With the Dot Plot you can look at two parameters at the same time.”

Question 20

What is Gating?

Answer 20

You can draw a ‘gate’ around any group of cells.

Question 21

How is Analysis done?

Answer 21

You can ask the computer to filter out cells depending on the stains.