Section 5: Flow Cytometry

Question 1

When was the automated cell counter developed?

Answer 1

in the 40s

Question 2

when was the Coulter cell counter developed?

Answer 2

in the 50s

Question 3

When was the first commercially available cell counter PDP 11

Answer 3

1969

Question 4

When was the first Coulter available?

Answer 4

1971

Question 5

Flow Cytometry

Answer 5

-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

Question 6

What are the clinical uses of flow cytometry?

Answer 6

-cell phenotyping (cancer cells, T cells of HIV patients, etc.)
- X-linked Hyper IgM syndrome (CD40 ligand- CD154)
- Allergy (basophil activation markers)

Question 7

What are the research uses of flow cytometry?

Answer 7

-in addition to cell phenotyping, cell sorting can be achieved using flow cytometry (FACS)

Question 8

Fluidics

Answer 8

-Sheath fluid is used to focus the sample so that a single cell passes through an aperture by the principles of laminar flow

Question 9

optics

Answer 9

-lasers, filters, and photodiodes are used to capture scattered and emitted light
-Forward scatter (FSC)- relative size
-Side Scatter (SSC) - granularity/ complexity
- fluorescence intensity

Question 10

electronics

Answer 10

-photodiodes convert light captured to electric signals and communicate the signals to the CPU

Question 11

Flow cytometry- Laminar flow

Answer 11

-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

Question 12

Flow cytometry- electronics

Answer 12

-convert analog signal to proportional digital signals
-interface with computers to display and analyze data

Question 13

Flow cytometry- (optics)

Answer 13

-the larger it is = the more forward scatter
-the more granular it is = the more side scatter it has `

Question 14

Flow cytometry (reading graphs)

Answer 14

-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

Question 15

fluorochrome

Answer 15

-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

Question 16

Characteristics of fluorochromes

Answer 16

-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

Question 17

fluorescence histogram

Answer 17

-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

Question 18

Flow cytometry- optical filters

Answer 18

-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

Question 19

flow cytometry- internal components

Answer 19

-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

Question 20

flow cytometry (procedure)

Answer 20

1. draw blood from subject
2. separate mononuclear cells using a ficoll gradient
3. Can either cryopreserve or stain with fluorescent antibody conjugates
4. optimize fluorescence detector for sensitivity
5. pass stained cell suspension through a laser beam
6. gate cell population of interest
   - ficoll is a protein that will migrate out and separate when you spin these red cells from white cells

Question 21

multiparameter flow cytometry

Answer 21

using multiple lasers and fluorochromes o identify a number of cellular
-TH17 is an instigator of inflammation

Question 22

flow cytometry- panel design

Answer 22

-an extremely important step to a successful multicolor flow experiment in the initial panel design

Question 23

What factors are involved in panel design?

Answer 23

-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)

Question 24

Gating

Answer 24

-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

Question 25

population frequencies

Answer 25

using population frequencies of specific cell types can be calculated
-CD45 indicates leukocytes and is expressed in them

Question 26

Flow cytometry (FACS)0 fluorescence assisted cell sorting

Answer 26

• here we can use a tag to identify some cell type