Section 5: Flow Cytometry Flashcards
When was the automated cell counter developed?
in the 40s
when was the Coulter cell counter developed?
in the 50s
When was the first commercially available cell counter PDP 11
1969
When was the first Coulter available?
1971
Flow Cytometry
-combines a direct or indirect immunofluorescent assay with cell sampling and optics system that allows the analysis of individual cells or particles from a sample
What are the clinical uses of flow cytometry?
-cell phenotyping (cancer cells, T cells of HIV patients, etc.)
- X-linked Hyper IgM syndrome (CD40 ligand- CD154)
- Allergy (basophil activation markers)
What are the research uses of flow cytometry?
-in addition to cell phenotyping, cell sorting can be achieved using flow cytometry (FACS)
Fluidics
-Sheath fluid is used to focus the sample so that a single cell passes through an aperture by the principles of laminar flow
optics
-lasers, filters, and photodiodes are used to capture scattered and emitted light
-Forward scatter (FSC)- relative size
-Side Scatter (SSC) - granularity/ complexity
- fluorescence intensity
electronics
-photodiodes convert light captured to electric signals and communicate the signals to the CPU
Flow cytometry- Laminar flow
-aperture pushed this fluid into this tiny stream which comes and drops through the aperture and gets hit with lasers and now we have detectors to give us a signal
Flow cytometry- electronics
-convert analog signal to proportional digital signals
-interface with computers to display and analyze data
Flow cytometry- (optics)
-the larger it is = the more forward scatter
-the more granular it is = the more side scatter it has `
Flow cytometry (reading graphs)
-when reading a cytogram (scatter plot) from a flow cytometry experiment focusing on the axes is important
-usually, only two parameters are examined on one graph
fluorochrome
-a fluorescent molecule that can emit light following excitation
-fluorochromes will absorb light (excited) in one wavelength and emit light (fluorescence ) in another
-the emitted light (fluorescent) is proportional to the number of binding sites present on the cell
Characteristics of fluorochromes
-maximum excitation and emission wavelength (nm)
-extinction coefficient- molar absorption of light
-quantum yield- efficiency of energy transfer
-lifetime: duration of excitation state
-stokes shift: the difference between max excitation and max emission wavelength
fluorescence histogram
-a histogram of fluorescence intensity is the easiest way to visualize these data
-a negative (lack target) will usually have peak events in the lowest range of the log fluorescence
-a positive (having target) will usually have a peak towards the higher end of the log fluorescence scale
Flow cytometry- optical filters
-the filters take out either the long pass ( these are the low wavelength values) to get it to be around 500 or use the short pass (these are higher wavelengths), or the band pass which is a little bit on each side
-the filter tries to remove some of the background or excess to try and make sure you have a nice clean signal
- ex: bandpass 50 +/- 50 means it goes from 450 to 550
-allow you to create a light source with different filters on the end
flow cytometry- internal components
-different kinds of light sources will hit certain fluorochrome differently. Some will excite and it can be read. So a red laser can read an Alexa or APC, side seven
-these conjugations of filters result in the detector giving options
-the money maker is the blue laser at 488 nm, a wide variety of different fluorochromes you can use
flow cytometry (procedure)
- draw blood from subject
- separate mononuclear cells using a ficoll gradient
- Can either cryopreserve or stain with fluorescent antibody conjugates
- optimize fluorescence detector for sensitivity
- pass stained cell suspension through a laser beam
- gate cell population of interest
- ficoll is a protein that will migrate out and separate when you spin these red cells from white cells
multiparameter flow cytometry
using multiple lasers and fluorochromes o identify a number of cellular
-TH17 is an instigator of inflammation
flow cytometry- panel design
-an extremely important step to a successful multicolor flow experiment in the initial panel design
What factors are involved in panel design?
-match the brightest fluorochrome with dimmest Ag (antigen density and intracellular antigens)
-choice combination that will minimize spectral overlap (compensation)
-choice and use of single stain controls (isotype controls, beads)
-negative controls (background fluorescent is controlled for depending on sample being used)
Gating
-when analyzing flow data Gaiting the samples is very important
-is a set value of limits or boundaries that are used to separate specific groups of cells
-is performed on a bivariate histogram and (scatter plot) that visualize two parameters at once
-when analyzing multiple parameters a gating strategy helps to visualize these cells in a way that is easy to interpret
-gates are drawn around clusters of specific cells or defined using a single parameter histogram and negative/isotype control
- lymphocytes, monocytes, and granulocytes separated by FSS and SCC
-lymphocytes separated by CD4 (y axis) and CD8 (x axis) expression
population frequencies
using population frequencies of specific cell types can be calculated
-CD45 indicates leukocytes and is expressed in them
Flow cytometry (FACS)0 fluorescence assisted cell sorting
- here we can use a tag to identify some cell type
