Flow cytometry Flashcards
What is flow cytometry?
= technique which simultaneously measures several physical characteristics belonging to a single cell in suspension - ‘in flow”
Done by light scatter and fluorescence
What is flow sorting?
= sorting cells based on properties measured in flow
- Also called fluorescence-activated cell sorting (FACS)
What does flow cytometry tell us?
- Relative size
- Relative granularity/internal complexity
- Relative fluorescence intensity
What are the three different stages in flow cytometry?
Fluidics
- cells in supension by injecting sample into a sheath fluid as it passes through a small orifice = hydrodynamic focusing (large volume into a small volume)
- flow in a single-file through an illuminated volume
Optics
- hit by a laser and scatter light and emit fluoresence
- single wavelength of light = laser line and provide coherent light
Electronics
- light signals are collected, filtered and converted to digital values
- stored on a computer
- analog-digital conversion
How does fluorescence happen?
When a laser hits fluorochrome and its excited at one wavelength, then goes back to unexcited state but emits fluoresecene at a higher wavelength…:
excitiation and emission spectrum
What is the stokes shift?
= energy difference between the lowest energy peak of absorbance and the highest energy of emission
What are the different types of fluorochromes and dyes?
Fluroescein isothiocyanate (FITC) = green
Phyocerythrin (PE) = orange
Peridinin chlorophyll protein = red
They all emit at different wavelengths
What are some ideal substances for flow cytometry?
- Peripheral blood
- Bone marrow
- Fine needle aspirate
- CSF and other fluids
- Fresh tissue
What are the different types of labelling for flow cytometry?
- Direct - monoclonal antibodies - proconjugated to fluorochromes
- Indirect - unconjugated monoclonal antibodies
How can flow cytometry be shown on graphs?
Histogram
- one-dimensional/one-parameter
- cell number Y-axis
- fluorscence X-axis
Dot plot
- two-dimensional/two-parameter
- side scatter X-axis and forward scatter Y-axis
How can you show 4 population cells on a dot plot?
What is gating?
Draw around the region on the dot plot you want to investigate on the dot plot
Then display only the inside on the gate on the basis of their fluorescence (compared to SS and FS originally
From gating, you can then analysis them using histogram and one parameter
- show cells that are negative and positive for each fluorescence
What is the purpose of propidium iodide?
- Used to detect cellular DNA by florescent dye
- undergoes a increase in fluorescence upon binding DNA
- requires permeabilization of the plasma membrane
Propidium iodide cannot normal cross the cell membrane
- If it does, it is assumed to be damaged
- So cells that are brightly fluorescent with PI are damaged or dead
What are the different detection methods of apoptosis for cells?
- Staining with dye PI (cells need to be fixed) - shows on histogram as Sub-G0 peak:
- Phosphatidyl serine normally is on the inside of the cell, but when the cell apoptosis, it flips so its on the outside. If you incubate cells with annexin V then it will detect if on the outside and therefore shows cells have apoptosis.
- Staining with 7-AAD (cells not fixed)
What is cell sorting?
- Gives analysis
- And lets us draw circles to isolate
