cell techniques and flow cytometry - mcgill...

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

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Examples of its uses

Surface marker expression

Cytoplasmic markers

intracellular concentrations of ions and ROS

DNA and cell cycle

Apoptosis

Hematopoietic cell development

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Mature

Hematopoietic Cell

Types

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Mature

Hematopoietic Cell

Types (2)

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Some prettier pictures of stained cells follow on the next 2 slides

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Lymphocytes are small when not activated. The nucleus is far more prominent than the cytoplasm

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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

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BloodSpleenLymph node

Bone marrowThymus

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Working with Mouse Tissues

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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!)

Counting Cells

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Haemocytometer

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.

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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.

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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)

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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

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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.

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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

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CD4 and CD8 T cells

intracellular cytokines

Ca++, pH, oxidative burst

karyotyping and chromosome sorting

cell cycle analysis

apoptosis

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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

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stream to about 20 micrometer dia.

Laser light focussed onto sample.

Light scatter. Group excitation.

Sorting - sometimes

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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

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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

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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

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For multicolour analysis

one needs to know the

excitation and emission

spectra of the

fluorochromes to use.

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Does the instrument

have 1 or 2 lasers?

What filters are

available?

Types of data display

Scatter Contour

Superimposed dots. No Like topographic

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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

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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.

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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.

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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

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pH Indicators e.g. SNARF family

A fluorescence

emission peak

wavelength shift

allows measurement

at two wavelengths

and a ratiometric

determination of

concentration

differences.

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differences.

Best excitation

wavelength would

be where all curves

(lower left) intersect

(about 530nm), but

not critical if

emission ratio

measured.

Intracellular pH

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Oxidative Burst

Dihydro-Rhodamine DHRMembrane permeable and non fluorescent.Converted to Rhodamine 123 by peroxide and peroxidase

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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

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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

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Bromodeoxyuridine Labelling for Cycle Analysis

Label in culture with BrdU.

Harvest, fix, permeabilize cells. Partially denature DNA. Incubate with Fluorescine-anti-BrdU.

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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

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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.

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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

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DNA fragmentation during apoptosis produces the kind of cytofluorimetric histogram of DNA staining shown right.

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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.