Flow Cytometry Flashcards
What technique is flow cytometry?
Technique which simultaneously measures several physical characteristics belonging to a single cell in suspension
What is FACS?
Sorting (separating) cells based on properties measured in flow
What does flow cytometry tell us?
FLOW CYTOMETER TELLS US: 1. Its Relative Size • Done without fluorescence 2. Its Relative Granularity/Internal Complexity • Done without fluorescence 3. Its Relative Fluorescence Intensity
What are the 2 methods of visualization?
- Fluorescence microscopy
2. Flow Cytometry
Fluorescence microscopy
- Intensity of cell is difficult to look at
* Less quantitative – would need to examine many fields
Flow cytometry
- Many cells in a flow cytometer – more quantitative
* Can look at intensity of each individual cell
How many parts if flow cytometry divided into and what are they?
• The way it works is divided into three parts:
○ Fluidics
○ Optics
○ Electronics
Basic steps of flow cytometry
- Cells in suspension
- Flow in single-file through illuminated volume where they scatter light and emit fluorescence
- Fluorescence is collected, filtered
- Converted to digital values
- Stored on a computer
In the fluidics part, how must the cells flow and how is this accomplished?
○ Must flow in single file
○ Accomplished by injecting sample into a sheath fluid as it passes through a small (50-300 µm) orifice
What is hydrodynamic focusing?
Introduction of a large volume into a smaller volume
In the optics part, what is the light source used?
Light source used is lasers
What do lasers hit and what are they scattered into?
Laser hits single file of cells and is scattered in two directions
What information do we get when laser is scattered in forward direction?
§ light that is scattered in forward direction = info about size of cell
What information do we get when laser is scattered at right angle?
§ right angle = granularity of cell
What happens in the electronics part?
Conversion of light signals to electrical signals
What is stokes shift defined as?
Stokes Shift: energy difference between the lowest energy peak of absorbance and the highest energy of emission
What are the 3 immunofluorescence proteins and their colors?
- Fluorescein isothiocyanate (FITC) – green
- Phycoerythrin (PE) - orange
- Peridinin Chlorophyll Protein – red
Why are 3 different immunofluorescence colors . used?
Three different colours used to measure three different parameters of the cells
What is used to differentiate between the 3 immunofluorescence colors?
○ Filters and mirrors can be used to differentiate between these colours where they overlap
What must we take into account when combining fluorochromes?
§ Must take into account overlap
What are 2 methods of labelling?
- Direct:Monoclonal antibodies(MoAbs) which are pre-conjugated to fluorochromes
- Indirect: Unconjugated MoAbs
Direct method of labelling
-one step method
What is the direct method of labelling helpful for?
○ Helpful for looking at many different antigens at the same time
Indirect method of labelling
-two step method
What are the 2 steps involved in indirect labelling?
- First step: add primary antibody
2. Second step: antibody to the antibody with the fluorochrome on it
What is the disadvantage of of indirect labelling?
Disadvantage: a lot of background staining with an indirect method
What are the 2 ways to display data of flow cytometry?
- Histogram
- Dot plot
How many parameters do histograms have?
One parameter
What are the axis in histograms?
○ X axis: fluorescence intensity
○ Y axis: cell count
How many parameters can you measure at a time with histograms?
-Can only measure one parameter at a time
What are the axis in dot plots?
○ X axis: side scatter
○ Y axis: forward scatter
How many parameters do dot plots measure?
○ Two parameters
§ Quantitate cells on basis of two parameters
§ Axis can be anything e.g. fluorescence
How can we display result on computer?
Gating
What is gating and what can we get from this?
• Using PC – can draw ‘gate’ around population
○ Can then get more info from the cells in this gate
i.e. what proportion of cells in the gate are CD+?
