Flow Cytometry

Question 1

What technique is flow cytometry?

Answer 1

Technique which simultaneously measures several physical characteristics belonging to a single cell in suspension

Question 2

What is FACS?

Answer 2

Sorting (separating) cells based on properties measured in flow

Question 3

What does flow cytometry tell us?

Answer 3

FLOW CYTOMETER TELLS US:
1. Its Relative Size
	     • Done without fluorescence
2. Its Relative Granularity/Internal Complexity
	• Done without fluorescence
3. Its Relative Fluorescence Intensity

Question 4

What are the 2 methods of visualization?

Answer 4

1. Fluorescence microscopy

2. Flow Cytometry

Question 5

Fluorescence microscopy

Answer 5

• Intensity of cell is difficult to look at

* Less quantitative – would need to examine many fields

Question 6

Flow cytometry

Answer 6

• Many cells in a flow cytometer – more quantitative

* Can look at intensity of each individual cell

Question 7

How many parts if flow cytometry divided into and what are they?

Answer 7

• The way it works is divided into three parts:
○ Fluidics
○ Optics
○ Electronics

Question 8

Basic steps of flow cytometry

Answer 8

1. Cells in suspension
2. Flow in single-file through illuminated volume where they scatter light and emit fluorescence
3. Fluorescence is collected, filtered
4. Converted to digital values
5. Stored on a computer

Question 9

In the fluidics part, how must the cells flow and how is this accomplished?

Answer 9

○ Must flow in single file

○ Accomplished by injecting sample into a sheath fluid as it passes through a small (50-300 µm) orifice

Question 10

What is hydrodynamic focusing?

Answer 10

Introduction of a large volume into a smaller volume

Question 11

In the optics part, what is the light source used?

Answer 11

Light source used is lasers

Question 12

What do lasers hit and what are they scattered into?

Answer 12

Laser hits single file of cells and is scattered in two directions

Question 13

What information do we get when laser is scattered in forward direction?

Answer 13

§ light that is scattered in forward direction = info about size of cell

Question 14

What information do we get when laser is scattered at right angle?

Answer 14

§ right angle = granularity of cell

Question 15

What happens in the electronics part?

Answer 15

Conversion of light signals to electrical signals

Question 16

What is stokes shift defined as?

Answer 16

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

Question 17

What are the 3 immunofluorescence proteins and their colors?

Answer 17

• Fluorescein isothiocyanate (FITC) – green
• Phycoerythrin (PE) - orange
• Peridinin Chlorophyll Protein – red

Question 18

Why are 3 different immunofluorescence colors . used?

Answer 18

Three different colours used to measure three different parameters of the cells

Question 19

What is used to differentiate between the 3 immunofluorescence colors?

Answer 19

○ Filters and mirrors can be used to differentiate between these colours where they overlap

Question 20

What must we take into account when combining fluorochromes?

Answer 20

§ Must take into account overlap

Question 21

What are 2 methods of labelling?

Answer 21

1. Direct:Monoclonal antibodies(MoAbs) which are pre-conjugated to fluorochromes
2. Indirect: Unconjugated MoAbs

Question 22

Direct method of labelling

Answer 22

-one step method

Question 23

What is the direct method of labelling helpful for?

Answer 23

○ Helpful for looking at many different antigens at the same time

Question 24

Indirect method of labelling

Answer 24

-two step method

Question 25

What are the 2 steps involved in indirect labelling?

Answer 25

1. First step: add primary antibody

2. Second step: antibody to the antibody with the fluorochrome on it

Question 26

What is the disadvantage of of indirect labelling?

Answer 26

Disadvantage: a lot of background staining with an indirect method

Question 27

What are the 2 ways to display data of flow cytometry?

Answer 27

• Histogram

- Dot plot

Question 28

How many parameters do histograms have?

Answer 28

One parameter

Question 29

What are the axis in histograms?

Answer 29

○ X axis: fluorescence intensity

○ Y axis: cell count

Question 30

How many parameters can you measure at a time with histograms?

Answer 30

-Can only measure one parameter at a time

Question 31

What are the axis in dot plots?

Answer 31

○ X axis: side scatter

○ Y axis: forward scatter

Question 32

How many parameters do dot plots measure?

Answer 32

○ Two parameters
§ Quantitate cells on basis of two parameters
§ Axis can be anything e.g. fluorescence

Question 33

How can we display result on computer?

Answer 33

Gating

Question 34

What is gating and what can we get from this?

Answer 34

• Using PC – can draw ‘gate’ around population
○ Can then get more info from the cells in this gate
i.e. what proportion of cells in the gate are CD+?