Light scatter
 Forward
angle light scatter
 in
a narrow angle from the direction of the laser
beam
 FALS or FS
 Right
 at
angle light scatter
right angles to the laser beam
 RALS or SS (side scatter)
Forward light scatter
FS
detector
laser
blocker bar
Light Scatter
 The
intensity of scatter is proportional to the
size, shape and optical homogeneity of cells
(or other particles)
 It is strongly dependent on the angle over
which it is measured
 particularly
with forward scatter
Light Scatter
 Forward
scatter tends to be more sensitive to
the size and surface properties
 can
be used to distinguish live from dead cells
 Side
scatter tends to be more sensitive to
inclusions within cells
 can
be used to distinguish granulated cells from
non-granulated cells
Gating
 Set
a region on a histogram or cytogram
 IF cell IN region THEN show another
property
Cell selection by gating
‘Gating’ on the lymphocytes. IF cell has light
scatter in R1 THEN show on CD4/CD8
cytogram
lymphocytes
Triggering the electronics
threshold
time
Changing the threshold setting
debris
Shape of the laser beam
Focus the laser beam with:
 spherical
lens - circular cross-section
 cross cylindrical lens pair - elliptical X-section
I
spherical
cross-section
elliptical
distance
Pulse shape analysis
flow
laser
Integrated area =
total fluorescence
signal
Signal peak
Signal width =
beam width + cell diam.
time
Pulse shape analysis
two cells
single cells
DNA analysis by flow
cytometry
Michael G. Ormerod
m.g.ormerod@btinternet.com
DNA content
Ploidy
Cell cycle
DNA Probes
Use DNA probes that are stoichiometric
- that is, the number of molecules of
probe bound is equivalent to the
quantity of DNA
Dyes for DNA cell cycle
analysis
 Propidium iodide
 Excited
at 488 nm; fluoresces red (617 nm)
 easily combined with fluorescein stain
 also stains RNA
 DRAQ5
 Max.
excitation at 646 nm; can be excited at
488 nm; fluoresces in deep red at 680 nm max
 Taken up by live cells
Dyes for DNA cell cycle analysis

Hoechst dyes
excited by uv; fluoresce blue
 DNA specific - bind to AT
 Hoechst 33342 can be used to stain viable cells


DAPI


excited by uv; fluoresce blue
DNA specific
Definitions & Terms
 DNA Ploidy
 Related
to the quantity of DNA in a cell
 DNA Index
 Ratio
between the mean DNA content of the
test cells to the mean DNA content of normal
diploid cells, in G0/G1phase
 Coefficient of Variation
 100*SD/mean
(CV)
DNA
 Usually measured on G1/G0 cells
DNA content - measuring ploidy &
SPF
Cell number
FNA of human breast carcinoma PI stain
G1
normal
tumour
S
DNA
G2
DNA analysis of the cell cycle
Following changes in the cell cycle
Quality control of DNA
measurement
 Sample
preparation
 Instrument alignment
 Correct data analysis
Using propidium iodide for
DNA analysis
 Excited
at 488 nm (argon-ion)
 Fluoresces red
 Does not cross intact plasma membrane
 Permeabilise
with detergent or
 Fix in 70% ethanol or
 Fix in paraformaldehyde followed by ethanol
 Treat
with RNase
Sample preparation for DNA analysis
 Fixed
cells
 Samples
can be stored
 Needed when adding antibody stain
 Quality may be reduced
 Permeabilisied cells
 Use
or nuclei
fresh or frozen samples, limited storage
time
 High quality achievable (Vindelov method)
DNA measurement
Use linear amplification
 Cell
cycle is linear, not logarithmic
 Relevant information occupies more of the
histogram
 Cell cycle algorithms assume a linear scale
Instrument alignment
 Check
daily using standard fluorescent
beads
 Correct alignment essential
 (Some misalignment can be tolerated with
immunofluoresence measurement - not
DNA)
DNA measurement
Use linear amplification
 Cell
cycle is linear, not logarithmic
 Relevant information occupies more of the
histogram
 Cell cycle algorithms assume a linear scale
Quality control of DNA measurement
Measure the spread of the distribution across
the G1/G0 peak as coefficient of variation
(cv)
DNA measurement
Human breast carcinoma cells prepared by the Vindelov
method. PI stain.
(Data supplied by Gyda Otteson & Ib Christensen, Finsen Laboratory, Copenhagen)
D
C T
C TD
A
C 1.2%
C, T cv = 1%
T 1.0%
D cv =D1.2%
1.0%
A 2.2%
DNA histogram
 Measure
DNA content
 Problem with clumps
 2 cells in G1 = 1 cell in G2
 Distinguish by pulse shape analysis
Shape of the laser beam
focus with:
 spherical
lens - circular cross-section
 cross cylindrical lens pair - elliptical crosssection
I
spherical
cross-section
elliptical
distance
Flow Cytometry
 Pulse
shape analysis
Integrated area =
total fluorescence
cell
beam
Signal peak
Signal width =
beam width + cell diam.
PMT
voltage
time
Pulse shape analysis
G1
G2
laser
2x
signal
width
area
ht
time
2 x G1
flow
Pulse shape analysis
DNA area
Pulse shape analysis
ungated
gated
single
DNA area
Measuring cell proliferation
using the BrdUrd/anti-BrdUrd
method
Measuring cell proliferation
 DNA histogram
 BrdUrd/anti-BrdUrd
 Hoechst/PI/BrdUrd
 Dilution
of label
DNA histogram
 Static
measurement of the cell cycle
 First choice
 Easy to combine with antibody stain
Following changes in the cell cycle
Cisplatin
S phase
slow down
Genotoxic
drug
G2 block
BrdUrd/anti-BrdUrd
 Pulse
label with BrdUrd (30 min)
 Harvest cells at different times
 Fix cells
 Denature DNA (acid, heat or UV)
 Label with anti-BrdUrd and PI
Cell cycle analysis
BrdUrd/anti-BrdUrd
V79 cells
(data supplied by G.
D. Wilson,, CRC
Gray Laboratories)
S
G2
G1
DNA/PI
BrdUrd/anti-BrdUrd
 Dynamic
analysis
 more complex procedure - denaturation of
DNA
 difficult to combine with another antibody
Exposure of the BrdUrd
 Denature
DNA with 2 M HCl or heat
 Partially digest DNA with
endonuclease/exonuclease
 UV irradiation - label strand breaks with
Tdt/BrdUrd (SBIP)
 Li
et al., (1994) Int. J. Oncol., 4, 1157.
 UV irradiation in
the presence of Hoechst
33258
 Hammers
et al. (2000) Cytometry, 40, 327.
Cell cycle analysis
BrdUdr/anti-BrdUdr
4h
0h
S
G1
G2
DNA/PI
8h
Measurement of proliferation
BrdUdr/anti-BrdUdr
0h
4h
8h
S
G1
G2
DNA/PI
V79 cells (data supplied by G. D. Wilson,, CRC Gray Laboratories)
BrdUrd/anti-BrdUrd
1
4
7
2
5
8
3
6
DNA
9
V79 cells
(data supplied
by G. D.
Wilson,, CRC
Gray
Laboratories)
Window set in early to mid-S
phase
Drug effects on cell cycle
pulse label after treatment
Incubated for 2 h with cisplatin 24 h earlier
No drug
Drug
Cells prepared in Institute for Cancer Studies, Sheffield
Nuclear & cytoplasmic antigens
Michael G. Ormerod
m.g.ormerod@btinternet.com
Staining intracellular antigens
 To
detect intracellular antigens, the cells
must be fixed or permeabilised.
 Method used depends on
 The
antigen to be detected
The combination of stains used in a multiparameter analysis
Staining intracellular antigens
 The
epitope on a particular antigen may be
sensitive to fixation
 Consequently, there is no standard
procedure for preparing cells
 A procedure has to be established for each
new antibody.
Fixatives for intracellular
antigens
 Fixatives
may be divided into two broad
classes
 Those
that cross-link proteins, such as
paraformaldehyde
 Those that coagulate proteins and extract lipids,
such as ethanol, methanol and acetone
 The
two may be combined - e.g.
paraformaldehyde followed by ethanol
Permeabilisation of cells
 Unfixed
cells can be permeabilised using a
variety of detergents. These can be divided
into two classes
 Strong
detergents, such as Triton-X 100, which
will dissolve the plasma membrane on unfixed
cells
 Weak detergents, such as saponin, which will
create holes in the plasma membrane
 Sometimes,
cells are fixed and then
permeabilised
Procedures for intracellular
antigens
 Typical
procedures include:
 Fixation
in 70% ethanol at 0°C
 Fixation in absolute methanol at - 20°C
 Fixation in 1% paraformaldehyde followed by
methanol, both at 0°C
 Incubation of fresh cells with antibody on ice in
the presence of detergent
 For nuclear antigens, enucleation with a strong
detergent followed by fixation.
Intracellular antigen + DNA
 Either:
 Fix
in 70% ethanol at 0°C, or
 Fix in 1% PFA followed by ethanol or
methanol
 Stain
with antibody-FITC
 Analyse
Human ovarian CA ascites
Labelled with antikeratin 8/18 & PI
tumour
diploid tumour?
normal
Fixed in PFA, methanol
Data supplied by W.E. Corver,
Leiden
Cyclins
cyclin A
FITC
control Ig
DNA
DNA
Molt-4 cells. Data supplied by Frank Traganos,
N Y.
Cyclin B
W1L2 human
lymphoblastoid
cells
Isolated nuclei
M
early
G1
S
G2
Ki-S1 (FITC)
Ki-S1 (FITC)
Ki-S1, proliferation-related antigen.
Nuclei from breast Ca cell line, ZR75.
G1
DNA
Data supplied by Richard Camplejohn
DNA
vinblastine treated
Two antigens plus DNA
 Fix
the cells
 1%
paraformaldehyde at 0°C followed by
methanol at -20°C
 Stain
for antigens using FITC & PE
 Stain for DNA
 Hoechst
3258
 7-AAD
 DRAQ5
 PI
+ TO-PRO-3
Cyclin B & p105 + Hoechst 33258
Prostate tumour cell line. Data supplied by James Jaccoberger.
mitotic cells
Endoreduplication
Further applications to cell and
molecular biology
Estimating cell viability
 Incubate with
fluorescein diacetate (FDA)
 Add propidium iodide (PI)
FDA
esterase
fluorescein
+ PI
fluorescein
dead
viable
Estimating cell viability
clumps
live
dead
PI
Ovarian Ca
cell line
labeled with
FDA & PI
Monitoring electropermeabilisation
 Electroporate at
0°C
 Add propidium iodide (PI)
 Warm to 37°C and incubate 10 min
 Add fluorescein diacetate & incubate 10
min
 Analyse green and red fluorescence
Monitoring electropermeabilisation
electroporate
+ PI
not
permeabilised
fail to
reseal
permeabilised
reseal
incubate 37°C add FDA