L5- Flow Cytometry

Question 1

What is it

Answer 1

Analysis of cell characteristics as they singly pass in a fluid suspension through a beam of light/laser beam

Question 2

What 4 characteristics can be determined

Answer 2

Size , granularity (fsc and ssc)

Cell components and function (through tagging)
Example is are they dividing? Cell cycle phase?

Question 3

How would you determine cell based on size

Answer 3

Forward scatter patterns

As light passes through, diffraction occurs and the larger the diffraction the larger the cell
Detected by a fs detector

Question 4

How is granularity detected to determine cell eg neutrophils

Answer 4

Side scatter patterns - this is at 90• angle from the laser beam
More side scatter means more granularity

Question 5

At whcih angle is fluorescence detected

Answer 5

90• too like side scatter

Question 6

Which 2 types of fluorescence methods are there

Answer 6

Fluorochrome tagged antibodies for specific proteins

Or fluorescent dyes

Question 7

What is 2 major examples of fluorochromes commonly used

Answer 7

PE and FITC

Question 8

How can you distinguish diff fluorochromes

Answer 8

Absorb and reemit light at specific wavelengths

Question 9

What are the detectors of fluorescence called and how many usually

Answer 9

PMTs photomultiplier tubes

Usually 4 as usually 4 fluorchromes used

Question 10

What are the 2 peaks/spectra at different wavelengths representing for fluorochromes

Answer 10

First peak is the wavelength is absorbs light (excitation spectra)
Second peak is the wavelength it emits light at (emission spectrum)

Question 11

What is the first filter light from fluorochromes Will pass through and why - determine which fluorochromes detected by which pmt

Answer 11

Dichroic mirror
Allows passage of particular wavelengths of light

Question 12

What are the filters called just before PMTs which allow passage of shorter wavelengths / specific before photon detection by pmt

Answer 12

Colour filters / BandPass filter

Question 13

What wavelength of light would a bandpass filter of 585/42 pass through

Answer 13

585 +- 21

Half the second number plus or minus

Question 14

Applications: 1. DNA ANALYSIS

For dna analysis, what fluorescence technique would be used

Answer 14

Intercalating dyes proportional to amount of dna
Eg propidium iodide

Question 15

What 3 things can you determine from dna analysis flow cytometry

Answer 15

Cell cycle phase
Dna index
Apoptotic cells

Question 16

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

Answer 16

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

Question 17

If you added a drug which arrests cells in g0/1 phase what would you see for cell count over dna content/fluorescence

Answer 17

Massive g1 peak and lack of g2 peak/ quite low cell count in general so no more peaks.

Question 18

How do you calculate dna index in cancer cells with abnormal

Answer 18

Dna content g0/1 peak of tumour cell

/ Typical standard 2N g0/1 peak (200 fluorescence)

Question 19

If there was a g1 peak at 300 fluorescence what does this indicate and what is the dna index

Answer 19

Hyperploidy

300/200 = 1.5

Question 20

What is a value of 0.5, more than 1 or less than 1

Answer 20

Haploid, hyper , hypo/ aneu

Question 21

Why is dna index important in the context of leukemia

Answer 21

Hypoploudy is associated with poorer prognosis

Question 22

Why is dna index important in breast and prostate cancer

Answer 22

Aneu is associated with relapse or poorer prognosis

Question 23

Looking at dna index also helps distinguish inflamed cells vs lymphoma cells. How

Answer 23

Inflamed won’t have anything but diploidy

Question 24

What is the cell count over fluorescence pattern for apoptotic cells

Answer 24

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

Question 25

Application 2: phenotypic analysis

Question 26

Why is phenotypic analysis important and how to do it

Answer 26

Can tag via antibodies uo to 4 different intracellular or cell surface antigens and you can see from the clustering pattern whether cells are positive or negative

Determine cells of hematopoietic lineage that can then be selected using facs

Question 27

What cancer has it been used to diagnose and identify minimal residual disease for

Answer 27

Acute and chronic leukaemias

Question 28

How would you go about looking at T cell proliferatice abilities using cell cycle and surface staining combined

Answer 28

Using cd3+ marker for example
and then also intercalating dye to study the normal cell cycle

Question 29

What would normal T cells cell cycle look like vs cancer/malignant

Answer 29

Just a peak at g1, they do not divide usually

Full cell cycle suggests malignancy

Question 30

What is the type of flow cytometry which aims to sort cells based on fluorescence markers or scattering patterns desired

Answer 30

Facs

Question 31

What is the most common way to separate them

Answer 31

Electrostatic separation using high voltage plates (- or + charge)

Question 32

Explain how it works

Answer 32

Nozzle which vibrates is added to disrupt the flow stream and form droplets

By the time cells reach this break point in the flow stream, a decision has been made on which charge to apply

The droplets are them charged and deflected to subsequent voltage plates
Eg cells with positive charge move towards -ve electrostatic plate