Applications of Flow Cytometry

Question 1

What is a major use of flow cytometry

Answer 1

Earliest applications of flow cytometry was the analysis of cell cycle position by quantitation of cellular DNA

Method of choice for fast, accurate determination of cell cycle distributions

Question 2

Describe the simplest univariate cell cycle method?

Answer 2

In the simplest method, cellular DNA is detected using a fluorescent dye that binds preferentially to DNA

Question 3

What is the fluorescent dye used in flow cytometry?

Answer 3

Propidium iodide (PI) is most commonly used

Undergoes dramatic increase in fluorescence upon binding DNA
PI emits ~600-620 nm

Question 4

How is PI able to access DNA to bind to?

Answer 4

It requires permeabilization of the plasma membrane as not able to cross membrane normally - manipulated to test for viability

Question 5

How is PI used to test cell viability for flow cytomery?

Answer 5

PI cant normally cross cell membrane

If PI penetrates cell membrane, its assumed to be damaged

Cells brightly fluorescent with PI are damaged or dead

Question 6

What is apoptosis?

Answer 6

Apoptosis is programmed cell death where the cell goes through a highly regulated process of “dying”

Question 7

What are the characteristics of apoptosis?

Answer 7

Condensation of chromatin

Blebbing of nuclear material

Often accompanied by internucleosomal degradation of DNA -> distinctive ‘ladder’ pattern on DNA gel electrophoresis

Question 8

How is apoptosis detected by flow cytometry?

Answer 8

By staining with PI dye (cells fixed)

Phosphatidylserine, detected by incubating cells with fluorescein-labelled AnnexinV, and PI (cells not fixed)

Staining with 7-aminoactinomycin D (cells not fixed)

Question 9

Describe the characteristics of apoptotic cells in cell cycle

Answer 9

Sub G0 peak seen during apoptosis

Question 10

Why is the sub G0 peak not a reliable detection of apoptosis?

Answer 10

Debatable whether these cells are debris or apoptotic cells

Cells undergoing apoptosis don’t always display this peak

Question 11

How are unviable apoptotic cells identified using staining?

Answer 11

1. Agent added into cells to initiate apoptosis
2. Stained with PI at various time points
3. Can see how G0 peak increases over time
4. Can add markers around any of the peaks to quantitate
   no. of cells →

Question 12

Describe the normal location of phosphatidylserine in cells

Answer 12

Phosphatidylserine normally on inside of a live cell

PI unable to enter live cells

Question 13

How does AnnexinV-FITC identify apoptotic cells?

Answer 13

AnnexinV-FITC directly conjugates to Phosphatidylserine which is now on outside of cells undergoing apoptosis

Question 14

How do we distinguish live cells?

Answer 14

Live cells test -ve for both PI and AnnexinV-FITC

Question 15

How are early apoptotic cells identified?

Answer 15

Early apoptotic cells +ve for AnnexinV-FITC but PI still can;t get in so still -ve for PI

Question 16

How does staining identify necrotic cells ?

Answer 16

In late apoptotic / necrotic cells, PI is able to enter as membrane is damaged, so now test +ve for both PI and AnnexinV-FITC

Question 17

What are the 3 populations visualised using the flow cytometry dyes?

Answer 17

Live cells - negative for both dyes
Dyed cells - positive for both
Apoptotic cells - annexin positive but PI negative

Question 18

Describe the fluorescence spectra for 7-aminoactinomycin D (7AAD)

Answer 18

Excitation: ~488 nm
Emission: ~660 nm (red)

Question 19

How does 7AAD bind to DNA?

Answer 19

DNA-specific - intercalates in G-C regions

Question 20

How can we use 7AAD along with other dyes?

Answer 20

long emission wavelength
can use with FITC & PE labelled Ab for simultaneous evaluation of DNA content and 2-color immunofluorescence using only 488 nm Ex

Question 21

What 3 characteristics can be identified using 7AAD?

Answer 21

7AAD gives 3 characteristic populations :

• Live cells -ve
• Apoptotic cells (dim)
• Dead cells +ve (show brighter)

Question 22

What further analysis can we do on cells treated with 7AAD?

Answer 22

Can purify the cell populations based on their staining intensity with 7AAD
Can measure any component of apoptosis cascade

Question 23

What are the applications of flow cytometry?

Answer 23

• Immunophenotyping of leukaemias & lymphomas
• Detection of MRD
• Stem cell enumeration
• CD4/CD8 in HIV
• Measurement of intracellular cytokines
• Study of cell cycle, viability & apoptosis
• Measurement of cell proliferation
• Assessment of transfection efficiency

Question 24

Describe the process of cell sorting

Answer 24

1. Cells introduced into vibrating nozzle tip
2. Laser hits cells in single file as they leave nozzle
3. Fluorescence emitted
4. Data displayed on CPU

Question 25

What is the difference between cell sorting and flow cytometry?

Answer 25

Cells in single file stay in flow for a certain time before breaking off due to the vibration

Question 26

What is the benefit of a vibrating nozzle tip in cell sorting?

Answer 26

Charges certain cells (that satisfy specific regions) to be detected by deflection plates into sorted fraction - can quantitate time between charged cells and can differentiate between 2 regions at once

Cells that don’t satisfy by region (specific) go to waste