Flow cytometry I - introduction

Question 1

What is flow cytometry?

Answer 1

Technique which simultaneously measures several physical characteristics belonging to a single cell in suspension by:
- Light scatter and fluorescence

Question 2

What is flow sorting?

Answer 2

Sorting 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 fluorescence intensity
• its relative granularity

Question 4

How does a flow cytometer get information about a cell?

Answer 4

It measures:

• Anything on the cell surface (receptors, adhesion molecules)
• intracellular cytokines or enzymes
• the DNA (cell cycle, apoptosis, viability of the cell)

Question 5

What is an other method of cell visualisation than flow cytometry

Answer 5

Fluorescence microscopy

Question 6

Difference between flow cytometer and fluorescent microscope

Answer 6

Fluorescent microscopy:
- only a limited view of cells - - hard to quantitate cells accurately (especially with the eye)

Flow cytometry:
-we are looking at thousands of cells every second

• the intensity of the fluorescence is quite variable and we make subjective judgements, whereas the flow cytometer it gives a very accurate view of how intense the fluorescence is

Question 7

Advantages of flow cytometry

Answer 7

· quick
· can look for rare cells
· quantifiable
· more accurate and quantifiable intensity

Question 8

Basics of Flow Cytometry Machine

Answer 8

· 3 components:

Fluidics
Optics
Electronics

Question 9

Requirements in fluidics

Answer 9

Cells need to be in suspension to flow in a single file

Question 10

How are the cells in suspension organised to flow in a single file?

Answer 10

Hydrodynamic Focusing

• injecting a sample fluid (cells) into central core
• this runs side by side to the sheath fluid (but they don’t mix), creating a Laminar flow
• sample cells pass through a small 50-300um orifice one by one at the junction where the analysis by lasers begins

Question 11

What happens after cells flow in a single file?

Answer 11

Optics
-the laser hits the cells one by one

-light is scattered in two directions which are picked up by detectors, obtaining cellular information (without even adding any antibodies or fluorescence yet)

Forward light scatter → proportional to the size of the cell

90° light scatter (side scatter)→ proportional to the granularity/internal complexity of the cell

Question 12

Features of lasers used in flow cytometry

Answer 12

• 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 13

What happens when laser light hits the cell

what is the wavelength of the laser light

Answer 13

• Light hits the cell and is scattered in a forward direction proportional to the size of the cell = forward light scatter
• Light is also scattered at a right angle to the cell proportional to granularity = side scatter

488nm

Question 14

What happens after light scatters are produced?

Answer 14

The different cell populations already distinguished are labelled with different antibodies with fluorochromes on them (immunofluorescence)

Laser→ fluorescence is emitted from cells, passes through filters and mirrors and we pick that up by the photo multiplier tubes (PMTs)

Question 15

Why are filters and mirrors needed?

Answer 15

We need the system of filters and mirrors because of the overlap in the emission spectrum of the different fluorochromes.

Mirrors are responsible for directing light through the detection path

Filters are placed in front of detectors to restrict light introduced to each PMT so that each detector is dedicated to measure fluorescence from a specific fluorochrome

Question 16

Role of photo multiplier tubes (PMTs)

Answer 16

Electronics

-convert the light (analogue) into digital

Question 17

How does fluorescence happen in flow cytometry?

Answer 17

Fluorescence happens when a fluorochrome is excited by a laser and then it goes back to its unexcited state, emitting fluorescence at a higher wavelength

Question 18

What is the Stokes shift?

Answer 18

The Stokes shift is the energy difference between the peak excitation and peak emission wavelengths.

Question 19

Fluorochromes and dyes commonly used in immunofluorescence

Answer 19

· Fluorescein isothiocyanate (FITC)→ emits green

· Phycoerythrin (PE)→emits orange

· Peridinin Chlorophyll Protein (PerCP)→emits red

*all excited by the common 488nm laser

Question 20

Why are FITC, PE and PerCP commonly used together in immunofluorescence?

Answer 20

Because they emit at different wavelengths we can detect them all at the same time. We can add in three different antibodies with different fluorochrome on them all at the same time to obtain information on the cells.

Question 21

Single cells in suspension for flow cytometry include:

Answer 21

Peripheral blood
Bone marrow
Fine needle aspirate
CSF and other fluids
Fresh tissue

Question 22

How do we label the cell with antibodies?

Answer 22

Direct Labelling

• monoclonal antibodies (MoAbs) are preconjugated to fluorochromes and then added to cell suspension
• wash and then put through flow cytometer

Indirect Labelling

• unconjugated MoAbs added to cell suspension
• wash
• then we add a secondary antibody with a fluorochrome on it to the primary antibody
• wash and then put through flow cytometer

Question 23

How to display data from a flow cytometer on a computer

Answer 23

Two ways of displaying data on the computer:

• Dot plot (looks at two parameters→e.g. forward scatter & side scatter)
• Histogram (looks at one parameter)

Question 24

How many populations can you identify with two fluorochromes?

Answer 24

Using our two-dimensional dot plot, we can identify four populations of cells, either:

• negative for both
• single positive for FITC
• single positive for PE
• double-positive for both

Question 25

Principle of flow cytometry data analysis

Answer 25

GATING
-creating ‘gates’ on the dot plot to distinguish cells of interest and analyse those specific cells

Then, analysis is used to quantitate the populations.

Question 26

How many populations can you identify with three fluorochromes?

Answer 26

If we increase that to three colours, we can now look at eight different populations of cells, either positive for all three, negative for all three or combinations in between. Therefore, three different fluorochromes will give you the ability to distinguish eight different populations.