Flow Cytometry Workshop

Question 1

Why is Flow cytometry used?

Answer 1

It is a high through put and high sensitivity method of identifying single cells of interest.

Question 2

What fields use flow cytometry?

Answer 2

1. Proteomics
2. Cytomics

Question 3

What are the roles of flow cytometry?

Answer 3

1. Multi-colour surface staining
2. Cell sorting or purification
3. Expression cloning

Question 4

What is expression cloning?

Answer 4

Enriching a very low frequency population and expanding them.

Question 5

What are the different types of flow cytometers?

Answer 5

1. Mobile to work in remote areas with remote populations like in Africa.
2. Static which are very expensive and large.
3. Multiple laser lines to detect more sub populations in one run.

Question 6

Define flow cytometry

Answer 6

Measuring properties of cells in a fluid stream.

Question 7

Define Fluorescence-Activated Cell Sorting

Answer 7

Sorting the cells based on the properties measured in the flow cytometry

Question 8

What can you measure with a flow cytometer?

Answer 8

1. Relative cell size and granularity
2. Fluorescence emission when dyes bound to cells are excited by the laser
3. Emission from many dyes at once for each cell
4. Data from thousands of cells per second which allows you to see subtle changes

Question 9

What happens in a flow cytometer?

Answer 9

1. The flow cell focuses the cell suspension to make a single line of cells.
2. A laser is shone at the cell line and we can get information about the cell from how the light scatters
3. We can get additional information about the cell by using fluorescent dyes and measuring the wavelength of light emitted.

Question 10

What does the forward scatter tell us?

Answer 10

The size of the cell

Question 11

What does the side scatter tell us?

Answer 11

The shape and granularity of the cell

Question 12

How is the scatter data visualised?

Answer 12

By converting the light into a voltage and then making a plot

Question 13

What do the fluorescent dye wavelength emissions tell us?

Answer 13

1. What dye is attached to the cell and therefore identify the cell
2. How much of the dye there is

Question 14

How are fluorescently labelled antibodies used in flow cytometry?

Answer 14

1. To identify the phenotype of receptors present on the cells you are measuring
2. Test large numbers of cells
3. Measure multiple characteristics by using multiple labelled antibodies
4. dissect complex cell populations
5. define rare populations
6. measure relative levels of antibody binding

Question 15

How can a cell sorter be used in flow cytometry?

Answer 15

1. It sorts cells of interest based on the fluorescent signals.
2. An electrical charge is applied to the cell and this sorts them onto charged plates.

Question 16

Flow cytometer data presentation: single parameters

Answer 16

On a histogram

Question 17

Flow cytometer data presentation: Bivariate data

Answer 17

Displayed in 2 dimensions using dot plots, density or contour plots

Question 18

Flow cytometer data presentation: 3 or more parameters

Answer 18

Displayed as sets of histogram or bivariate plots or using cluster analysis

Question 19

How is gating used in flow cytometry?

Answer 19

1. To detect rate cell populations like stem cells.
2. To remove dead cells from the data
3. To prevent false positives
4. To identify any unwanted cells

Question 20

What is MFI?

Answer 20

Mean fluorescence intensity which is the comparison of brightness of different cell populations or samples

Question 21

What are the applications of flow cytometry?

Answer 21

1. Analysis of surface or intracellular protein expression
2. Resolution of complex populations

Question 22

What are the applications of Flow sorting?

Answer 22

1. Isolation of individual subpopulations from complex mixtures of cells
2. Cloning specific cell populations

Question 23

What are fluorescent tracking dyes used for?

Answer 23

Measuring cell proliferation

Question 24

How do fluorescent tracking dyes work?

Answer 24

1. They are cell permeable and covalently couple to intracellular proteins.
2. Can identify up to 8 cell divisions as the signal halves with every division

Question 25

What makes a flow cytometer a sorter?

Answer 25

The ability to select any population defined by a logical combination of regions and isolate this population from the sample.

Question 26

How is flow cytometry used in gene expression studies?

Answer 26

1. To purify cell populations before microarrays
2. Post gene-chip-confirm protein expression using reporters

Question 27

What are the uses of flow cytometry and nucleic acid stains?

Answer 27

The purpose of these is to measure the DNA content.
1. Cell cycle analysis
2. Measure ploidy
3. DNA probes