flow cytometry

Question 1

what is flow cytometry

Answer 1

technique which simultaneously measures several physical characteristics belonging to single cell in suspension

1. done by light scatter fluorescence

Question 2

define flow sorting

Answer 2

sorting cells based on properties measured in flow

also called fluorescence activated cell sorting

Question 3

what does a flow cytometer tell us about a cell

Answer 3

1. its size
2. its relative granularity/internal complexity
3. its relative fluorescence intensity

Question 4

what are the methods of visualisation

Answer 4

fluorescence microscopy

flow cytometry

Question 5

basics of flow cytometry

Answer 5

cells in suspension

flow in single file through

an illuminated volume where they scatter light and emit fluorescence

collected, filtered and converted to digital values and stored on computer

Question 6

fluidics

Answer 6

need to have cells in suspension flow in single file

accomplished by injecting sample into sheath fluid as it passes through small orifice

sample fluid flows in central core that doesn’t mix with sheath fluid - laminar flow

introduction of large volume into small volume - hydrodynamic focusing

Question 7

optics - light sources

Answer 7

lasers =
single wavelength of light or mix of wavelengths

can provide from milliwatts to watts of light

can be inexpensive, air cooled units or expensive, water cooled units

provide coherent light

Question 8

describe electronics

Answer 8

processing of signals from detectors = analog digital conversion

Question 9

fluorescence - stokes shift

Answer 9

energy difference between lowest energy peak of absorbency and highest energy of emission

Question 10

immunofluorescence: fluorochromes and dyes

Answer 10

FITC = green

phycoerythrin = orange

peridinin chlorophyll protein = red

Question 11

flow cytometry: single cell suspension

Answer 11

peripheral blood
bone marrow 
fine needle aspirate 
CSF and other fluids 
fresh tissue

Question 12

methods of labelling: immunofluorescence

Answer 12

direct = monoclonal antibodies are preconjugated to fluorochromes

indirect = unconjugated moABs

Question 13

univariate cell cycle methods

Answer 13

cellular DNA is detected using fluorescent dye that binds preferentially to DNA

propidium iodide = most commonly used. undergoes dramatic increase in fluorescence upon binding DNA.
requires permeabilisation go plasma membrane

Question 14

PI - cell viability

Answer 14

how assay works

1. PI cannot normally cross cell membrane
2. if PI penetrates cell membrane, it is assumed to be damaged
3. cells that are brightly fluorescent with PI are damaged or dead

Question 15

measurement of apoptosis

Answer 15

apoptosis = programmed cell death where the cell goes through highly regulated process of dying

characteristics are condensation of chromatin material

blebbing of nuclear material

often accompanied by internucleosomal degradation of DNA giving rise to distinctive ladder pattern on DNA gel electrophoresis

Question 16

detection methods for apoptosis

Answer 16

by staining with dye PI

phosphatidyl serine can be detected by incubating the cells with fluorescein-labeled annexing V, and PI

by staining with 7-aminoactinomycin D

Question 17

7-aminoactinomycin D

Answer 17

Ex: ~488 nm
Em: ~660 nm
DNA-specific
 intercalates in G-C regions
long emission wavelength 
with FITC & PE labeled Ab for simultaneous evaluation of DNA content and 2-color immunofluorescence using only 488 nm Ex

Question 18

applications of flow cytometery

Answer 18

immunophenotyping of leukaemia and lymphomas

detection of MRD

stem cell enumeration

CD4/CD8 in HIV

measurement of intracellular cytokines

study of cell cycle, viability, apoptosis

measurement of cell proliferation

assessment of transfection efficiency