1. reticulocyte analysis live/dead identify microorganisms
DNA & RNA Parameters
total DNA & RNA content
nucleic acid sequence
cell cycle analysis
chromosome analysis
reticulocyte analysis
live/dead
membrane integrity
identify microorganisms
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2. Spectra of PI and EtBr PI Ethidium 457 350 514 610 632 488 6:57 PM
600 nm
300 nm
500 nm
700 nm
400 nm
457
350
514
610
632
488
note that can excite EtBr at both and at to get the red fluorescence
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3. Mithramycin, Olivomycin, Chromomycin A3
Attach at G-C region of DNA
do not intercalate; need Mg++ to bind
Quantum efficiency low
Ex: ~440 nm Em: nm
Can excite with:
457 Ar line
441 HeCd line
436 Hg arc lamp line
GC vs AT for others
cells must be fixed, permeabilized
no interaction with RNA
Crissman and Hirons note 2 Ex peaks: ~320 & ~445
olivomycin Em chromomycin Em mithramycin Em 575
can use with Ar 457 though HeCd 441 or Hg arc 436 better
350
575
488
425
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4. DNA-specific when bound at AT
Hoechst
DNA-specific when bound at AT
bind to sequences of 3 AT pairs
bind to outer groove of DNA
do not intercalate
Ex: ~350 nm (UV) Em: ~460 nm (blue)
300 nm nm nm nm
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5. Cyanine Dyes Thiazole orange, thiazole blue, thioflavin T and others
Stain both RNA and DNA
Quantum efficiency greatly increased when bound to NA
very low when unbound
Cross membranes of intact cells
will also enter mitochondria
thioflavin T used as reticulocyte stain (Ex: Em: )
base selectivity not established?
also thiazole orange--developed protocol for use as reticulocyte stain in clinical flow cytometer (FACScan) that had a fixed 488 Ar laser
Ex: Em: 533
thiazole blue--Ex: ~ Em:
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6. Reticulocyte Analysis
150
150
112
112
RMI = 34
RMI = 0
Count
Count 75 75 37 37 .1 1 10
100
1000 .1 1 10
100
1000
log Thiazole Orange
log Thiazole Orange
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7. Cyanine Dyes TOTO-1 , YOYO-1, TOTO-3
developed to have high binding affinity and high quantum efficiency
homodimers of
thiazole orange, oxazole yellow, thiazole blue
positively charged side chains
do not penetrate intact membranes
not DNA-specific
treat cells with RNase
Shapiro suggests may have AT preference
(base selectivity still not established)
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8. Acridine orange Metachromatic To differentiate DNA from RNA
green intercalated between base pairs
excitation at ~488 emission at ~525
red stacked on RNA or ss DNA
excitation at ~457 emission at ~630
To differentiate DNA from RNA
selectively denature dsRNA, not DNA
stringent conditions ([AO] and ionic strength)
can measure total cellular RNA
500
600
700
800
picture is emission spectra only
acid-treat to selectively denature RNA
quantum efficiency actually decreases when bound to NA; the fluorescence is slightly quenched
under a microscope, fluorescence/phosphorescence of bound dye bleaches more rapidly than free dye so background decreases before bound...does not happen in flow cytometer so much background
green fluorescence of AO bound to DNA bleaches before red fluorescence/phosphorescence of AO bound to RNA
(lifetime of emission from RNA 15nsec so longer than just fluorescence)
the bright nuclear staining get with AO must be due to its increased concentration in the nucleus
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9. Chromosome Analysis (Bivariate Flow Karyotyping - porcine)
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10. Dual Staining of Cells Nuclear probes c-myc, c-fos, p53 monoclonal Ab
Cytoplasmic protooncogene probes
‘ras’, ‘neu’ monoclonal Ab
Cell surface antigens
p-glycoprotein
Breast carcinoma
identify ploidy with PI
identify epithelial origin with
cytokeratin antibody
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11. s Normal Cell Cycle G2 M G0 G0 - G1 400 1000 75 150 225 300 Cell Count
200
400
600
800
1000 75
150
225
300 G0 G0
- G1 M G0 G2 G1 s
Cell Count G2 M s 2N 4N
DNA Content
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12. © 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt
DNA analysis – using PI
Other basic fluorescence applications
Cell #
The basis for this working is that there is a relationship between the number of molecules of PI and the binding to DNA is stoichiometric– thus we can generally relate dye concentration to the amount of DNA
Intensity
© J. Paul Robinson, Purdue University Lecture0004.ppt
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13. Analyzing the DNA Histogram12

14. Use of Archival Material for DNA Flow
David Hedley J Histochem Cytochem 1983;31:
Use formaldehyde-fixed, paraffin embedded blocks
Allows retrospective study of patient populations with known outcome
Slide: Supplied by David Hedley
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15. Comparison of Fresh vs Embedded: Same Tumour
Original Sydney series
Surgical biopsies - one piece mechanically disaggregated with triton X-100 in medium - remainder fixed in formaldehyde, and processed through to paraffin blocks
Used DAPI as DNA stain, on ICP-22 flow cytometer.
Slide: Supplied by David Hedley
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16. intracellular calcium intracellular glutathione oxidative burst
Functional Assays
intracellular pH
intracellular calcium
intracellular glutathione
oxidative burst
phagocytosis
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17. Phagocytosis
FITC-Labeled Bacteria
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18. Cellular Functions Cell Viability Ionic Flux Determinations
Phagocytosis
Organelle Function
mitochondria, ER
endosomes, Golgi
Oxidative Reactions
Superoxide
Hydrogen Peroxide
Nitric Oxide
Glutathione levels
Ionic Flux Determinations
Calcium
Intracellular pH
Membrane Potential
Membrane Polarization
Lipid Peroxidation
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19. Fluorescent Indicators
How the assays work:
Superoxide: Utilizes hydroethidine the sodium borohydride reduced derivative of EB
Hydrogen Peroxide: DCFH-DA is freely permeable and enters the cell where cellular esterases hydrolyze the acetate moieties making a polar structure which remain in the cell. Oxidants (H2O2) oxidize the DCFH to fluorescent DCF
Glutathione: In human samples measured using 40 M monobromobimane which combines with GSH by means of glutathione-S-transferase. This reaction occurs within 10 minutes reaction time.
Nitric Oxide: DCFH-DA can indicate for nitric oxide in a similar manner to H2O2 so care must be used. DAF is a specific probe available for Nitric Oxide
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20. PI - Cell Viability How the assay works:
PI cannot normally cross the cell membrane
If the PI penetrates the cell membrane, it is assumed to be damaged
Cells that are brightly fluorescent with the PI are damaged or dead
Viable Cell Damaged Cell PI PI PI PI PI PI PI PI PI PI PI PI PI PI
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21. Ionic Flux Determinations
Calcium Indo-1
Intracellular pH BCECF
How the assay works:
Fluorescent probes such as Indo-1 are able to bind to calcium in a ratiometric manner
The emission wavelength decreases as the probe binds available calcium
Time (Seconds) 36 72
108
144
180
Stimulation
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8 50
100
150
200
Ratio: intensity of 460nm / 405nm signals
Time (seconds)
Flow Cytometry Image Analysis
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22. Phosphorylation assays
Phospho antibodies
Measure the phosphorylation state of intracellular proteins at the single cell level
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