Lecture 2 - Flow Cytometry

Question 1

What is flow cytometry

Answer 1

Test that allows rapid measurement of several parameters of a single cell as it moves through a beam of light

Question 2

Detectors of a flow cytometer

Answer 2

Forward scatter (detects cell size) Side scatter (detects cell granularity) Fluorescence detector (detects colour of emitted light)

Question 3

What ferries cells into laser beam?

Answer 3

Fluidics system

Question 4

What does each dot on a flow cytometry dot plot represent?

Answer 4

A single cell

Question 5

Populations commonly seen in a blood sample

Answer 5

Three populations: Lymphocytes Monocytes Granulocytes

Question 6

Answer 6

blood 2

Question 7

Uses of fluorochromes 1) 2) 3) 4)

Answer 7

1) Direct conjugates 2) Indirect conjugates (secondary labelling) 3) DNA labelling 4) Cytoplasmic dyes

Question 8

How is fluorescence used in flow cytometry? 1) 2) 3)

Answer 8

1) Attached fluorescent tag emits light when stimulated with a particular wavelength. 2) There is a wavelength which results in the greatest light emission (peak). 3) Detect EG: +/-30nm either side of peak for a particular fluorochrome

Question 9

What is spectral overlap?

Answer 9

Where the emission spectra of two different fluorochromes overlap. Is detected by the wrong detector

Question 10

How is spectral overlap compensated for?

Answer 10

Need to subtract FITC from PE

Question 11

Uses of flow cytometry 1) 2) 3) 4) 5) 6) 7)

Answer 11

1) Cell surface molecule expression 2) Intracellular molecule expression 3) Cell function 4) DNA content cycle and analysis 5) Apoptosis 6) Antigen-specific cell function 7) Cell sorting

Question 12

5 x 10^6 Splenocytes are stained with an:-CD4 FITC and an:-CD8 PE. 15% are CD4+ cells and 10% are CD8+ cells. How would a forward scatter vs side scatter dot plot look?

Answer 12

Question 13

5 x 10^6 Splenocytes are stained with an:-CD4 FITC and an:-CD8 PE. 15% are CD4+ cells and 10% are CD8+ cells. How would a FITC vs empty channel dot plot look?

Answer 13

Question 14

5 x 10^6 Splenocytes are stained with an:-CD4 FITC and an:-CD8 PE. 15% are CD4+ cells and 10% are CD8+ cells. How would a PE vs empty channel dot plot look?

Answer 14

Question 15

5 x 10^6 Splenocytes are stained with an:-CD4 FITC and an:-CD8 PE. 15% are CD4+ cells and 10% are CD8+ cells. How would a FITC vs PE dot plot look?

Answer 15

Question 16

5 x 10^6 Splenocytes are stained with an:-CD4 FITC and an:-CD8 PE. 15% are CD4+ cells and 10% are CD8+ cells. How would a histogram comparing the relative amount of unstained to FITC stained cells look?

Answer 16

Question 17

How can the number of cell divisions be measured using flow cytometry?

Answer 17

Carboxyfluorescin succinyl ester injected into cells. Splits evenly between daughter cells. With each generation, CFSE fluorescence decreases.

Question 18

What is CFSE?

Answer 18

Carboxyfluorescing succinyl ester. Binds to cytoskeletal proteins, begins emitting light. Divides evenly between daughter cells.

Question 19

How is CFSE detected?

Answer 19

Using FL-1 FITC detector

Question 20

Why can’t FITC and CFSE be used together?

Answer 20

CFSE emits at a very similar wavelength to FITC

Question 21

How would a CFSE vs empty channel dot plot look?

Answer 21

Question 22

How would a CFSE vs empty channel histogram look?

Answer 22

Question 23

How can specific T cell responses be measured with flow cytometry?

Answer 23

1) Tetramer staining 2) Cytokine staining

Question 24

Tetramer staining 1) 2) 3)

Answer 24

1) Four peptide-loaded MHCI/MHCII/CD1d bound to streptavidin via biotin. 2) Fluorescent molecules directly conjugated to tetramer 3) This detects T cells expressing TCR specific to MHC/CD1d and antigen

Question 25

Why bind MHC to streptavidin as a tetramer?

Answer 25

A single MHC won’t bind to TCR with sufficient avidity to be detected in flow cytometry

Question 26

MHCI tetramer

Answer 26

Heavy chain binds peptide, then bound to beta microglobulin. Biotinylated. Binds to CD8 T cell

Question 27

MHCII tetramer

Answer 27

Alpha and beta chains bind tetramer. Biotinylated. Binds to CD4 T cell

Question 28

What is used as a control for tetramer flow cytometry?

Answer 28

An empty tetramer, to screen for non-specific interaction

Question 29

What would a flow cytometry dot plot of an empty tetramer vs a dot plot of a loaded tetramer look like?

Answer 29