Flow Cytometry - Introduction

Question 1

What is flow cytometry?

Answer 1

It’s a 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’s the difference between flow cytometry and flow sorting?

Answer 2

FLOW CYTOMETRY: - measuring properties of cells in flow

FLOW SORTING:

• 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

What can we the relative fluorescent intensity be used for?

Answer 4

It can be used to look at different characteristics of the cell, such as:

• cell surface receptors and antigens
• adhesion molecules
• levels of intracellular cytokines and enzymes
• DNA

Question 5

What are some ways to visualise fluorescent cells?

Answer 5

• fluorescence microscopy

- flow cytometry

Question 6

Briefly, summarise how a flow cytometer works.

Answer 6

FLUIDICS: where cells in a suspension flow in a single-file

OPTICS: the cells flow through an illuminated volume where they scatter light and emit fluorescence

ELECTRONICS: the fluorescence is collected, filtered and converted to digital values that are stores on a computer

Question 7

Describe the fluidics part of flow cytometry.

Answer 7

You need to have the cells in suspension flow in single file.

This is accomplished by injecting a sample into a sheath fluid as it passes through a small (50-300 µm) orific
The sample fluid flows in a central core that does not mix with the sheath fluid - making it laminar flow.

The introduction of a large volume into a small volume - this is called hydrodynamic focusing.

Question 8

Describe lasers as the light source in the optics part of a flow cytometer.

Answer 8

It is a single wavelength of light (a laser line) or (more rarely) a mixture of wavelengths

• it can provide from milliwatts to watts of light
• can be inexpensive, air-cooled units or expensive, water-cooled units
• they provide coherent light (single frequency)

Question 9

How do we get information from the way light scatters when it hits the cell?

Answer 9

When the light hits the cell and it scatters, it scatters in two directions:

• forward light scatter, which is proportional to the size of the cell
• 90° light scatter, which is proportional to the granularity of the cell

Question 10

Describe the electronics part of flow cytometry.

Answer 10

It is where the processing of signals from detectors takes place.

It is the analog-digital conversion.

Question 11

What is Stokes Shift?

Answer 11

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

Question 12

List some common fluorochromes.

Answer 12

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

Question 13

What are some examples of samples of single cells in suspension?

Answer 13

SINGLE CELLS IN SUSPENSION

• peripheral blood
• bone marrow
• fine needle aspirate
• CSF and other fluids
• fresh tissue

Question 14

What are the two methods of labelling in immunofluorescence?

Answer 14

DIRECT : Monoclonal antibodies (MoAbs) are preconjugated to fluorochromes

INDIRECT: Unconjugated MoAb

Question 15

List an advantage and a disadvantage of using the indirect method of labelling.

Answer 15

(+) you get an amplified signal

(-) you get lots of background staining

Question 16

What are two common ways of displaying data, and a feature of each?

Answer 16

The two common ways of displaying data are:

• a HISTOGRAM: where you can measure 1 parameter at a time
• a DOT PLOT: where you can quantify cells based on 2 parameters