Applications of Flow Cytometry Flashcards
What is a major use of flow cytometry
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
Describe the simplest univariate cell cycle method?
In the simplest method, cellular DNA is detected using a fluorescent dye that binds preferentially to DNA
What is the fluorescent dye used in flow cytometry?
Propidium iodide (PI) is most commonly used
Undergoes dramatic increase in fluorescence upon binding DNA
PI emits ~600-620 nm
How is PI able to access DNA to bind to?
It requires permeabilization of the plasma membrane as not able to cross membrane normally - manipulated to test for viability
How is PI used to test cell viability for flow cytomery?
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
What is apoptosis?
Apoptosis is programmed cell death where the cell goes through a highly regulated process of “dying”
What are the characteristics of apoptosis?
Condensation of chromatin
Blebbing of nuclear material
Often accompanied by internucleosomal degradation of DNA -> distinctive ‘ladder’ pattern on DNA gel electrophoresis
How is apoptosis detected by flow cytometry?
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)
Describe the characteristics of apoptotic cells in cell cycle
Sub G0 peak seen during apoptosis
Why is the sub G0 peak not a reliable detection of apoptosis?
Debatable whether these cells are debris or apoptotic cells
Cells undergoing apoptosis don’t always display this peak
How are unviable apoptotic cells identified using staining?
- Agent added into cells to initiate apoptosis
- Stained with PI at various time points
- Can see how G0 peak increases over time
- Can add markers around any of the peaks to quantitate
no. of cells →
Describe the normal location of phosphatidylserine in cells
Phosphatidylserine normally on inside of a live cell
PI unable to enter live cells
How does AnnexinV-FITC identify apoptotic cells?
AnnexinV-FITC directly conjugates to Phosphatidylserine which is now on outside of cells undergoing apoptosis
How do we distinguish live cells?
Live cells test -ve for both PI and AnnexinV-FITC
How are early apoptotic cells identified?
Early apoptotic cells +ve for AnnexinV-FITC but PI still can;t get in so still -ve for PI
How does staining identify necrotic cells ?
In late apoptotic / necrotic cells, PI is able to enter as membrane is damaged, so now test +ve for both PI and AnnexinV-FITC
What are the 3 populations visualised using the flow cytometry dyes?
Live cells - negative for both dyes
Dyed cells - positive for both
Apoptotic cells - annexin positive but PI negative
Describe the fluorescence spectra for 7-aminoactinomycin D (7AAD)
Excitation: ~488 nm
Emission: ~660 nm (red)
How does 7AAD bind to DNA?
DNA-specific - intercalates in G-C regions
How can we use 7AAD along with other dyes?
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
What 3 characteristics can be identified using 7AAD?
7AAD gives 3 characteristic populations :
- Live cells -ve
- Apoptotic cells (dim)
- Dead cells +ve (show brighter)
What further analysis can we do on cells treated with 7AAD?
Can purify the cell populations based on their staining intensity with 7AAD
Can measure any component of apoptosis cascade
What are the applications of flow cytometry?
- 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
Describe the process of cell sorting
- Cells introduced into vibrating nozzle tip
- Laser hits cells in single file as they leave nozzle
- Fluorescence emitted
- Data displayed on CPU
What is the difference between cell sorting and flow cytometry?
Cells in single file stay in flow for a certain time before breaking off due to the vibration
What is the benefit of a vibrating nozzle tip in cell sorting?
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
