cell techniques and flow cytometry - mcgill...
TRANSCRIPT
Cell Techniques and Flow Cytometry
Cells of the immune system
Cell preparations and fractionations
Functional assays
The flow cytometer and cell sorter
Examples of its uses
February 2008 [email protected] 1
Examples of its uses
Surface marker expression
Cytoplasmic markers
intracellular concentrations of ions and ROS
DNA and cell cycle
Apoptosis
Hematopoietic cell development
February 2008 [email protected] 2
February 2008 [email protected] 3
Mature
Hematopoietic Cell
Types (2)
February 2008 [email protected] 5
Some prettier pictures of stained cells follow on the next 2 slides
February 2008 [email protected] 6
Lymphocytes are small when not activated. The nucleus is far more prominent than the cytoplasm
February 2008 [email protected] 7
The Megakaryocyte is a very large cell. They are the factories for producing Platelets which bud off from this cell bone-marrow cell.
Lymphoid Tissues
Many cells of the immune system can be obtained easily in single cell suspension for study in vitro.
Blood
February 2008 [email protected] 8
BloodSpleenLymph node
Bone marrowThymus
February 2008 [email protected] 9
Working with Mouse Tissues
February 2008 [email protected] 10
Kill, clean, cut, pull skin away to reveal thoracic cage and peritoneal cavity, open the cavity, remove tissue sterilely, disrupt the tissue by cutting, teasing and pressing. (This picture actually shows embryos not spleen!)
Preparing and Fractionating Cells
Removal of debris and dead cells
Removal of red blood cells by lysis
These methods are dealt with in the notes which will be
available from http://www.endolab.mcgill.ca/courses/610B/
Some have been covered by another lecturer.
February 2008 [email protected] 12
Removal of red blood cells by lysis
Gradient sedimantation - e.g. Ficoll
Adherent cells - e.g. macrophage, dendtritic cells
T cell enrichment
Negative selection with antibody and complement
Panning on antibody coated plates
Magnetic beads
Some Common Types of Functional Assay
Proliferation
3H Thymidine incorporation (DNA synthesis)
Dye uptake (cytoplasmic volume, or enzyme level)
Again, these methods are dealt with in the notes, and some
have been covered by another lecturer.
February 2008 [email protected] 13
Dye uptake (cytoplasmic volume, or enzyme level)
Cytokine secretion
Cytotoxicity
Chromium release
Colourimetric (e.g. LDH release)
Haemolytic Plaque Assay for Ab-producing B cells
Red Blood Cells as Indicators of Ab and Ab-producing cells
Surface of rbc can be coated with antigen
Ab binds to Ag
complement then causes rbc lysis(serum from other animal
e.g. guinea pig serum sheep red blood cells)
protein via chromic chloride (mechanism unclear)
February 2008 [email protected] 14
Ab binds to Agsheep red blood cells)
Ab-producing cell
In agar on slideor in monolayer
Slide chamber for haemolytic plaque assay in agar support
February 2008 [email protected] 15
Reverse Haemolytic Plaque Assay
o Protein A coated on erythrocytes (often sheep — RBC)
o Specific Ab bound to protein A
o Sensitized rbc plus Ag-secreting cells form monolayer attached to poly-L-lysine-coated floor of chamber (allows washes and medium changes)
o Complement lyses cells with Ag bound to Ab.
February 2008 [email protected] 16
From: Boockfor and Fidan (2004) Methods 33: 273–280
o Complement lyses cells with Ag bound to Ab.
In this example, pituitary cellsare incubated with rbc sensitized with Ab specific for prolactin
FACS – a preparative and analytical tool
Forward and side light scatter
Single colour fluorescence histogram
Fluorochromes excitation and emission spectra
considerations for multi-colour analysis
Examples
CD4 and CD8 T cells
February 2008 [email protected] 17
CD4 and CD8 T cells
intracellular cytokines
Ca++, pH, oxidative burst
karyotyping and chromosome sorting
cell cycle analysis
apoptosis
February 2008 [email protected] 18
The Flow CytometerWhere it all comes together
Cells pumped into flow cell, and enter faster flow of sheath fluid.
Laminar flow conditions dynamically reduce the sample stream to about 20 micrometer
February 2008 [email protected] 19
stream to about 20 micrometer dia.
Laser light focussed onto sample.
Light scatter. Group excitation.
Sorting - sometimes
February 2008 [email protected] 20
February 2008 [email protected] 21
Sid
e S
ca
tte
r p
uls
e h
eig
ht
Light Scatter
This alone can discriminate several subpopulations and can allow gating for data acquisition from
February 2008 [email protected] 22
Sid
e S
ca
tte
r p
uls
e h
eig
ht
Forward Scatter pulse height
data acquisition from one of them
Dead cells and debris can be avoided
Stained Cell Fluorescence
If only one cell surface marker is being analysed, the primary antibody need not carry a fluorochrome tag.
Number
February 2008 [email protected] 23
tag.
A second (tagged) Ab may be used.
Data can be displayed simply as a histogram
Fl-1 peak height
Same analysis on different instruments
may show different sensitivities
February 2008 [email protected] 24
For multicolour analysis
one needs to know the
excitation and emission
spectra of the
fluorochromes to use.
February 2008 [email protected] 25
Does the instrument
have 1 or 2 lasers?
What filters are
available?
Types of data display
Scatter Contour
Superimposed dots. No Like topographic
February 2008 [email protected] 26
3D projection
Superimposed dots. No
idea of height. Merging
of subpopulations
Like topographic
map. Indication of
height.
Good visual
representation especially
for presentation. Not
useful for gating.
Example:
Leukocytes
T cells
stained with Ab
to:
CD3 (part of T Gated on medium forward, low side scatter sub-population
February 2008 [email protected] 27
cell receptor
complex)
CD4 (helper)
CD8 (cytotoxic)
Gated on medium forward, low side scatter sub-population
B cells express
none of these
markers.
Cytokines - Inside the cells
Fix and
permeabilize
before
incubation
with
antibodies.
February 2008 [email protected] 28
antibodies.
See notes for
how.
Loading cells with indicators
Some dyes will enter the cell and bind to specific cell components like DNA.
Some do not pass through the intact membrane, but can be esterified into a form which does.
February 2008 [email protected] 29
esterified into a form which does.
AM = acetoxymethyl -
Removed by non-specific esterases in the cell. Thus trapped. (But see notes).
This example is fura-2, a Ca++
probe
Fluo-3 as indicator of intracellular Ca++ concentrations
Indo-1 and Fura-2 are other commonly used calcium ion probes
February 2008 [email protected] 30
pH Indicators e.g. SNARF family
A fluorescence
emission peak
wavelength shift
allows measurement
at two wavelengths
and a ratiometric
determination of
concentration
differences.
February 2008 [email protected] 31
differences.
Best excitation
wavelength would
be where all curves
(lower left) intersect
(about 530nm), but
not critical if
emission ratio
measured.
Intracellular pH
February 2008 [email protected] 32
Oxidative Burst
Dihydro-Rhodamine DHRMembrane permeable and non fluorescent.Converted to Rhodamine 123 by peroxide and peroxidase
February 2008 [email protected] 33
February 2008 [email protected] 34
Gating on single cells using peak shape
Cells stained for DNA content.
Area of the fluorescence pulse is related to amount of DNA. (1N doublet similar to 2N
February 2008 [email protected] 35
Peak Width
doublet similar to 2N single cell)
Width is related to time for the particle to pass through the LASER beam. (1N doublet up to 2x as long)
1 n
2 n (or 2x 1n)
3 n (1n+2n)
s phase
probably
dead cells
doublets
The improvement by gating singlets
February 2008 [email protected] 36
Bromodeoxyuridine Labelling for Cycle Analysis
Label in culture with BrdU.
Harvest, fix, permeabilize cells. Partially denature DNA. Incubate with Fluorescine-anti-BrdU.
February 2008 [email protected] 37
Also stain DNA with PI.
Only those cells undergoing DNA synthesis will be labelled during the BrdU pulse.
1 n 2 ns
Cell Cycle Kinetics by BrdU Pulse and Chase
Labelled in s-phase chased through mitosis. Now BrdU labelled 1n
Brd
U i
nco
rpo
rate
d in
to D
NA
du
rin
g S
-ph
ase
February 2008 [email protected] 38
Cells not labelled during the pulse entering s-phase
Brd
U i
nco
rpo
rate
d in
to D
NA
du
rin
g S
stained DNA content
Labelling via TdT (terminal deoxynucleotidyl transferase reaction)
Staining to measure BrdU incorporation for data in the previous slide was by partially denaturing the DNA to expose BrdU epitope for a specific antibody to this nucleotide analogue. But this reduces binding of the dye which stains double-stranded DNA.
Another method relies on the breakage of DNA strands at BrdU sites upon exposure to UV light.
February 2008 [email protected] 39
upon exposure to UV light.
The DNA ends generated in this way can be labelled using the enzyme TdT, which can add radio-, fluorescent-, biotin-, or digoxygenin-labelled deoxynucleotides to the ends of DNA.
This method is also used to show DNA fragmentation in cells undergoing apoptosis.
I did not manage to talk of apoptosis in class. But please
read notes and be aware of the use of Anexin and TdT
February 2008 [email protected] 40
February 2008 [email protected] 41
DNA fragmentation during apoptosis produces the kind of cytofluorimetric histogram of DNA staining shown right.
February 2008 [email protected] 42
Apoptotic cell phosphatidyl-serine flips from inner to outer membrane. Annexinebinds to it.
Unlike necrotic cells, live and early apoptoticcells exclude the dye propidium iodide which stains the DNA.