L5- Flow Cytometry Flashcards
What is it
Analysis of cell characteristics as they singly pass in a fluid suspension through a beam of light/laser beam
What 4 characteristics can be determined
Size , granularity (fsc and ssc)
Cell components and function (through tagging)
Example is are they dividing? Cell cycle phase?
How would you determine cell based on size
Forward scatter patterns
As light passes through, diffraction occurs and the larger the diffraction the larger the cell
Detected by a fs detector
How is granularity detected to determine cell eg neutrophils
Side scatter patterns - this is at 90• angle from the laser beam
More side scatter means more granularity
At whcih angle is fluorescence detected
90• too like side scatter
Which 2 types of fluorescence methods are there
Fluorochrome tagged antibodies for specific proteins
Or fluorescent dyes
What is 2 major examples of fluorochromes commonly used
PE and FITC
How can you distinguish diff fluorochromes
Absorb and reemit light at specific wavelengths
What are the detectors of fluorescence called and how many usually
PMTs photomultiplier tubes
Usually 4 as usually 4 fluorchromes used
What are the 2 peaks/spectra at different wavelengths representing for fluorochromes
First peak is the wavelength is absorbs light (excitation spectra)
Second peak is the wavelength it emits light at (emission spectrum)
What is the first filter light from fluorochromes Will pass through and why - determine which fluorochromes detected by which pmt
Dichroic mirror
Allows passage of particular wavelengths of light
What are the filters called just before PMTs which allow passage of shorter wavelengths / specific before photon detection by pmt
Colour filters / BandPass filter
What wavelength of light would a bandpass filter of 585/42 pass through
585 +- 21
Half the second number plus or minus
Applications: 1. DNA ANALYSIS
For dna analysis, what fluorescence technique would be used
Intercalating dyes proportional to amount of dna
Eg propidium iodide
What 3 things can you determine from dna analysis flow cytometry
Cell cycle phase
Dna index
Apoptotic cells
When you look at cell count AND dna content combined pattern. What should be the pattern seen of fluorescence (X) i.e dna content 2N/4N and then on y axis cell count
Highest peak for cell count is g0/g1 phase
Which is also at 2N / 200 is usual standard of fluorescence here
Drop in cell count and plateau at S phase moving up to 400/ 4N on x axis
Smaller g2/m peak for cell count at 400/4N and that is where cell cycle would stop
If you added a drug which arrests cells in g0/1 phase what would you see for cell count over dna content/fluorescence
Massive g1 peak and lack of g2 peak/ quite low cell count in general so no more peaks.
How do you calculate dna index in cancer cells with abnormal
Dna content g0/1 peak of tumour cell
/ Typical standard 2N g0/1 peak (200 fluorescence)
If there was a g1 peak at 300 fluorescence what does this indicate and what is the dna index
Hyperploidy
300/200 = 1.5
What is a value of 0.5, more than 1 or less than 1
Haploid, hyper , hypo/ aneu
Why is dna index important in the context of leukemia
Hypoploudy is associated with poorer prognosis
Why is dna index important in breast and prostate cancer
Aneu is associated with relapse or poorer prognosis
Looking at dna index also helps distinguish inflamed cells vs lymphoma cells. How
Inflamed won’t have anything but diploidy
What is the cell count over fluorescence pattern for apoptotic cells
There is a sub g1 peak-
A peak before g1 at 2N/200 because durinf apoptosis dna is broken down and released from cells
Meaning lack of dye intercalation
