L8-Flow cytometry Flashcards
(29 cards)
What does flow cytometry depends upon ?
Fluorescent molecules. Different fluorescent molecules may be excited by different wavelengths of light
and emit different wavelengths.
What is a flow cytometer and what does it do?
-A machine that analyses particles (normally cells) in suspension
It will:
1.Count the particles
2.Tell you how big/granular they are
3. Detect fluorescent markers on the particles
How did flow cytometry arised?
- Antibody technology
- Sensitive light detection technology
– that can detect different brightness levels
3.Increasing computer sophistication - (Biologists being lazy
What more information can flow cytometry can give you ?
-What type of cells you have – how many and of what “markers” they express
-What the cells are up to
-Quantification of rare populations
-Track changes over time
-Break down heterogenous populations of cells into individual populations
What is you cells needs to be in for flow cytometry? otherwise it wont work
cells need to be in a suspension
Flow cytometry CAN’T tell you :-
-Cell location in your original sample
-What other cells/features do they sit next to in the tissue?
-Information about a single cell
(flow cytometry is all about populations).
What type of cells can we study from flow cytometry ?
-Normally: Cells – bacteria, eukaryotic cells
-With fancy machines:
Organelles, chromosomes
Viruses
Organisms: Drosophila embryos, C. elegans
However, the cells/particles must be in suspension!
What are the features that flow cytometry have? like the equipment
-laser
-cells in single file
-mirrors separating wavelengths of light
-computer for data in real time
-detectors
***Flow cytometers detect light and turn it into electronic signals
To detect size in F.C. what is used and how is it done?
Forward scatter
-Light bends (refracts) when moving through different densities (e.g. your straw in a glass)
-So when it enters the cell it refracts and then light beam becomes wider.
-Larger cell = larger width = larger electronic signal
More light in forward scatter means …
larger electronic signal
What does it mean when light bounces off objects inside a cell ?
-In this case, light still passes through my blackcurrant squash (which we see on the wall)
-Some of the light hits the ice and bounces off in all directions (which my camera detects)
What is used to detect granularity
side scatter - detects how much stiff is in it
What does combining data from size and granularity tells us?
1 = large and granular
2 = medium size and granularity
3 = small and not very granular at all
Clouds of cells are called what?
population
How to use fluorescent dye and antibodies in identifying things?
We can identify different cells through the molecules they have on their outside/inside
Put a different fluorescent molecule on each antibody
Antibodies will stick to their unique molecule
You will detect the fluorescence and be able to detect that cell
How to detect T cells
T cells all have a T cell receptor
There are two main types of T cells – helper (CD4) and cytotoxic (CD8)
Use antibodies to the T cell receptor to detect T cells then, antibodies to CD4 and CD8
How to detect different antibodies using many filters for different channels?
The more antibodies you use,
the more fluorochromes you need to detect,
the more filters you need
- Mixed wavelengths of light and other wavelengths reflected off, and selected wavelengths pass through.
Brighter the cell the larger the…
electronic signal
How are the data analysed in flow cytometry
All the data on size, granularity and each fluorescence channel is collected simultaneously.(at the same time)
Normally it is analysed sequentially e.g. identify the right size, then broad markers, then specific ones.
We use regions (or gates) to select cells and analyse them further
What do we use for high parameter data ?
(super high parameter data - when we don’t know what to expect - is analysed by computer algorithm)
Flow cytometry measures 2 changes in terms of cell numbers
-increasing cell numbers >migration>proliferation
-decreasing cell numbers >migration out> cell death
How to track how cell numbers change with time?
Count the number of cells you have of each type at different times.
In this example mice having an immune response were studied at the start, and then days 7, 21, 28 and 35 of an infection.
How to measure proliferation
Using a dye that can enter cells but never leave:
-Fluorescent molecule enters cells and an ester group is cleaved off.
-It can’t leave the cell.
-The fluorescence is halved during cell division
e.g. Traditionally: Carboxyfluorescein
succinimidyl ester (CFSE)
How do T cells proliferate in infection?
CFSE stained T cells transferred into mouse
Mouse vaccinated
Proliferation checked at day 3
