Download 3rd seminar (FACS) 2016

• Preparative and analytical methods based
on antibody-antigen interactions
• Functional investigations of the cells of the
immune system
(1st part)
The characterisation and separation of the cells of the
immune system
Flow cytometry (FACS)
The sensitivity of immunoassays
Many cell types of the immune system are indistinguishabe by
simple morphology (e.g. lymphocyte subtypes)
The most important cell surface molecules (”antigens”) of the
lymphocyte subtypes
Cell surface molecules – as markers – can be used for identifying cell types
The ligands of the cell surface receptors would be used to
label the cells
Problems:
• affinity
• large scale manufacturing
CD40
CD40L
CD40
CD40L
Cell surface antigen specific antibodies can be easily
produced
CD40
anti-CD40
The affinity/avidity of the antibodies can be appropriate for the purpose
To recognise the antigens on the cells, the label should
be also detectable
The antibodies should be also “visualised”
possibilities:
• isotopes
• enzyme (e.g. colour change catalysed by the enzyme)
• fluorescence
• microparticles (electron microscopy – “immunogold”)
• …etc…etc…
• …
Amino acid side chains of the proteins can be simply coupled to
labels with reactive chemical groups 1.
e.g. to amines on lysine side chains:
isothyocyanate group (…ITC)
succinimidyl ester (…SE) group:
Amino acid side chains of the proteins can be simply coupled to
labels with reactive chemical groups 1.
e.g. to thiols on cysteine side chains:
with haloalkalnes (alkyl halides):
with maleimide groups:
Fluorescent labels/dyes used in
flow cytometry or microscopy
The ideal fluorescent labels:
• photostable  slow fading
• narrow excitation and emission spectra  multiple dyes can
be used together
Fluorescent spectra internet tools:
http://www.bdbiosciences.com/us/s/spectrumviewer
http://www.biolegend.com/spectraanalyzer
https://www.thermofisher.com/hu/en/home/life-science/cell-analysis/labelingchemistry/fluorescence-spectraviewer.html
https://www.chroma.com/spectra-viewer
Fluorescent dyes
Fluorescein
excitation
emission
FITC (fluorescein isothiocyanate)
fluorescein-5-isothiocyanate
2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)-5isothiocyanatobenzoic acid
Lots of isomers exist with slightly different fluorescent spectra!
Fluorescent dyes
Phycoerythrin, PE (protein)
3Z-phycoerythrobilin (PEB),
the chromophore group of the
phycoerythrin protein
Phycoerythrin is an accessory pigment in different algae.
Helps the function of the main chlorophyll pigments
responsible for photosynthesis.
FLOW CYTOMETRY
An immunofluorescent method that mutually
complements the fluorescent microscopy
Large part of the method is using monoclonal antibodies
Detection and analysis of different cells or particles
travelling at high velocity in flow
Detects fluorescence intensity and scattered light of the
labeled cells
Can give indirect morfological data (size, granularity)
Statistical method:
Can investigate enormous number of cells in a short
period of time
ADVANTAGES OF THE FLOW
CYTOMETRY IN IMMUNOLOGY
Most cells in the immune system can be found in free or loosely adherent
form. They can be easily dispersed and labeled by fluorescent antigen
specific antibodies, and then they can be examined cell by cell in
suspension
The cells’ light scatter and immunofluorescent properties can be analyzed
statistically (e.g. percentages of different cell populations)
Rare cell populations can be identified and examined (e.g. antigen specific
lymphocytes)
The method provide qualitative and quantitative data – it can detect the
presence of different antigens in the cell, and the expression levels of these
antigens. Changes in the expression of certain molecules can be followed
after different treatment of the specimen. (e.g. cell activation, disease
progression)
CHARACTERIZATION OF IMMUNE CELLS
USING CELL SURFACE MARKERS
Cell types, differentiation stages can be identified using a
combination of cell surface markers.
Used in diagnostics:
- ratio of different cell types
- altered expression of cell surface markers
Examples:
- Inflammatory processes – increased neutrophil numbers
- HIV progression – decrease of CD4+ T cell count
CD4+ : CD8+ = ~1.6
Normal CD4+ T cell count = 600 – 1400/l
AIDS = CD4+ T cell count <200/l, altered CD4+ : CD8+ ratio
- increase of CD5+ B cells – typical for some B cell leukemias
Some typical markers of the main immune cell types
CD antigen
cell types
function
ligand
CD3
T cells
T cell antigen receptor
signalling chain complex
CD4
helper T cells, plasmacytoid dendritic cells (pDC), monocytes
co-receptor of the T cell
antigen receptor, (also HIV
entry receptor)
MHC II, (HIV)
CD5
T cells, (B cell sub-population: B1)
cell adhesion, signalling (costimulation)
CD72
CD8
cytotoxic T cells, (NK, some  T
co-receptor of the T cell
antigen receptor
MHC I
cells)
CD14
Monocytes, macrophages,
some granulocytes
part of the LPS signalling
receptor complex
LPS, LBP
CD19
B cells
part of the B cell Ag receptor
co-receptor complex
C3d, C3b
(CD19/CR2(CD21)/CD81)
CD28
T cells
co-stimulation
(B7-1, B7-2)
CD80, CD86
CD34
hematopoietic progenitor cells,
endothelial cells
cell adhesion, …
CD62L
(L-selectin)
CD56
NK cells, (some T and B cell sub-
homoadhesion (N-CAM
isophorm)
population)
CD80, CD86
(B7-1, -2)
professional APC: DC, B,
monocytes, macrophages
co-stimulation, cell adhesion
CD28, CD152
Some picture from the flow cytometers’ history: From the
first cell-counting machines to the high speed cell sorters
©J.Paul Robinson
Early cell counter. Katherine Williams and C.S. Sanders (Atomic Energy
Research Establishment) 1948 - Unclassified in 1956. (Photo taken in
Science Museum, London UK)
(cytometers from the 2000s)
benchtop flow cytometer
(BD FACSCalibur™)
sorter flow cytometer
(BD FACSDiVa™)
benchtop sorter flow cytometers from the recent ages
BD FACS Aria™
Beckman Coulter MoFlo™ XDP
FACS (acronym)
• original meaning: Fluorescence Activated Cell Sorting
• nowadays generally: flow cytometry
Basic principles of the flow cytometry
The cells could be rapidly and easily investigated in
laminar flow
a: The erythrocytes are arranged
in the laminar flow stream and
travel in elongated manner by the
hydrodynamic forces
b: In turbulence the cells are
disoriented, scattered and they are
travelling with different velocity
in the flow
Image fromV. Kachel, et al.
Hydrodynamic focusing
in the laminar flow
The ink is arranged to a
thin stream in the laminar
flow
There is no mixing
between the ink and the
sheath fluid
The original position of
the ink have only slight
influence on the position
of the thin stream in the
laminar flow
V. Kachel, H. Fellner-Feldegg & E. Menke
The hydrodynamic system of the flow cytometers
sheath fluid reservoir
Flow cell
injector
+++
+++
+++
sheath
fluid
sample
6-10m/s velocity
Fluorescent
signals
Focused laser
beam
Measured parameters:
Light scattering (FSC, SSC)
Any particle can
scatter the light
FALS Sensor
FSC
Laser
(forward angle
light scatter or
forward scatter)
The FSC has some
correlation with the
particle size
90 (side) scatter
SSC (side scatter)
Smaller structures, organeles inside the cells
effectively scatter the blue light in this way
Measured parameters:
Fluorescence
Laser
FSC
High sensitivity photon detectors (photomultipliers)
(photoelectron multiplier tubes – PMT)
detection of the fluorescence:
fluorescent dyes (and autofluorescence by the presence of pyridines and flavins)
The scheme of a flow cytometer
detectors
dicroic mirrors
and filters
PMT 4
sample
PMT 3
flow cell
PMT 2
FSC
detector
PMT 1
Laser(s)
IMMUNOPHENOTYPING BY FACS
Example:
Measurement of CD4+ (helper) and CD8+ (cytotoxic) T cell ratio
(e.g. monitoring AIDS progression)
Labeling:
FITC labeled anti-CD4 antibody(α-CD4-FITC)
PE labeled anti-CD8 antibody (α-CD8-PE)
Th
NK
Tc
Lymphocytes in the peripheral blood sample
Fluorescent
microscopy
B
high velocity flow stream
detecting CD4-FITC
labeled (TH) cell
(in cuvette or stream in air)
detector
signal
processing unit
CD8
PE
screen
increasing
light intensity
a dot representing a
CD4+ CD8- cell
CD4
FITC
microscopy:
detecting the PE labeled cell
(CD8-PE)
CD8
PE
detector
signal
processing unit
increasing
light intensity
CD4
FITC
detecting the unlabeled cell
(e.g. B cell) by autofluorescence
CD8
PE
detector
Signal
processing unit
increasing
light intensity
microscopy:
dim (autofluorescent)
cell
CD4
FITC
CD8
PE
18%
44%
animation!
0%
quadrant
statistics
CD4 38%
FITC
GRAPHICAL REPRESENTATIONS 1.
dot-plot
contourplot
densityplot
GRAPHICAL REPRESENTATIONS 2.
Histogramm
Numeral intensity
values (MFI):
~7
~ 1300
homogenous cell
population is normally
distributed (Gaussian)
Different cell types - characteristic light scattering
granulocytes
side light scattering
(SSC)
(e.g. granulated)
monocytes
lymphocytes
forward light
scattering (FSC)
(„size”)
EXAMINATION OF PERIPHERAL BLOOD BY HAEMATOLOGY AUTOMATS
Measured parameters:
peroxydase staining (the presence of myeloperoxydase, x – axis)
light scatter (high on large granular cells, y – axis)
1 (Noise)
2 Nucleated Red Blood Cells
3 Platelet Clumps
4 Lymphocytes and Basophils
5 Large Unstained Cells (LUC)
6 Monocytes
7 Neutrophils
8 Eosinophils
The major cell types can be easily identified
by the help of only two parameters!
The "gating" helps to identify the properties of the
cell populations
lymphocytes
granulocytes
all cells
granulocyte gate
monocytes
monocyte gate
limfocyte gate
Are they really CD4+ or it is autofluorescence?
Autofluorescence or real expression?
In the case of the lymphocytes the CD4 staining is obvious:
There were CD4+ (positive) and CD4- (negative) populations
CD4-
CD4+
But there are more difficult cases:
e.g. CD1a, MHC-like molecule expression on dendritic cells
Where are the CD1a positive cells?
Comparison with an unstained sample
?
(or with an “isotype control” stained sample)
human CD1a specific mouse IgG1 atibody
“control” mouse IgG1 antibody
Where to draw the border?
?
It is rewarding to draw the specidic fluorescence
together with the autofluorescence:
IgG1 isotype CD1a
control antibody
A control sample stained with isotype
control antibody
No staining can be seen within the
area where the specific staining will
be expected
The histogram “hides”
this cells
autofluorescence
autofloureszcencia
The histogram representation could be
misleading
Cell Biology / Immunology
The cellular functions can be also examined
by flow cytometry with fluorescent indicators, the so called
fluorescent “probes”:
•
•
•
•
•
•
DNA content
intracellular ion concentrations
redox state
pH
organelle number
…etc…
(They will be discussed on the next lecture also)
(Cell biology methods)
Investigation of the cell function
The intensity of the cell metabolism can be characterised by the numbers
of the mitochondria. Functional mitochondrion specific "probes" (e.g.
Mitotracker dyes) can be used for this purpose, which can make
distinction between living and dead cells also.
The nucleic-acid content of the cells can be measured by fluorescent dyes
that bind stoechiometrically to the nucleic-acids. Such kind of double
stranded nucleic-acid (DNA, dsRNA) incorporating stains are:
•propidium jodid
•ethidium bromid
Propidium
NH2
Ethidium
NH2
NH2
MitoTracker
Red
CH
N+
2
C2H2)3
N+
NH2
N+
5
CH3
C2H5
C2H5
The size of the cycling cells are increased –
called blast transformation
Cell-cycle
Possibilities of the examination
Stimuli
(e.g. antigen)
resting lymphocyte
(G0)
effector cell
- transcription (RT-PCR)
- protein synthesis
memory cell
(Immunoassay)
changes in the RNA- and protein
synthesis, in the cell membrane
and in the transports
cell division
change in the
number of the cells
DNA-synthesis
(MTT, CFSE)
DNA quantification
(fluorescent DNS intercalating
agents, 3H-thymidine)
The cell cycle can be examined by fluorescent dye
G2
G0
M
that intercalates stoechiometrically
into the double stranded DNA
(e.g. propidium iodide, PI)
DNA analysis
G1
cell number
s
G0G1
G2 M
s
0
200
400
600
800
1000
4N
2N
DNA content
Distribution of a normal cycling cell-population by
DNA content (flow cytometry)
DNA Analysis
A typical DNA Histogram
sub G0/G1
cells
0
200
400
2N
600
800
4N
PI Fluorescence
1000
Cell cycle specific protein detection can be used for the
examination of dividing cells:
Ki-67, PCNA, modified cyclins – can be shown by antibodies
Apoptosis detection:
•DNA loss during apoptosis
sub G0/G1
cells
•Inter nucleosomal cleavages result free
DNS ends which can be enzymatically
(Tdt) labeled by BrDU or dUTP
normal cycling cells
•Apoptotic changes in the
plasmamembrane (extracellular
appearance of the intracellular
phosphatidylserine) can be detected by
fluorescent dye conjugated Annexin V
protein
Chromosome analysis and separation
Chromosomes from cell cultures can be
investigated (and separated) by flow cytometry.
Two fluorescent dyes are used:
Hoechst 33258 binds the AT-rich regions,
and the chromomycin A3 binds the GC-rich
regions in the DNA.
Because of the different AT/GC ratios of the
chromosomes, they can be discriminated
Separated chromosomes, chromosome pools are
useful in the genomics
Hoechst 33258
Normal human
Human X hamster
Normal hamster
Normal mouse
Chromomycin A3
J.W. Gray & L.S. Cram
Kinetic measurements by flow cytometry
(the time can be also measured as a parameter)
In homogenous cell population the consecutive
measurement of some property in different cells is
represent the change of that property:
measurement
in one cell
measurement in
homogenous cell
population
The intracellular Ca2+ signal can be measured with
fluorescent Ca2+ indicators:
e.g. Fluo-3 or Indo-1
Single cell microscopic kinetic measurement:
Ca2+ cell signalling
Intracellular Ca2+ signal in one cell:
B cell
Antigen presenting B cell activates
a T cell which is preloaded with a
Ca2+ indicator dye
T cell
e.g. Fluo-3
or
Indo-1
Fluorescence intensity
representing the intracellular
Ca2+ concentration
Intracellular Ca2+ signal measurement by flow cytometry
activation
of the cells
time
base signal
(the time can be also measured as a parameter)
Intracellular Ca2+ signal measurement by flow cytometry
Ca2+ signalling in Influenza virus hemagglutinin protein derived peptide specific
T cell hybridome induced by the peptide pre-loaded antigen presenting cells
activated T cells
(no measurement during the
cell activation: the APCs and
the T cells are centrifuged to
close together)
non-activated T cells
Imaging cytometry
(More detailed morphologic information – sophisticated picture analysis softwares)
CIRCULARITY:
cell differentiation
cell division (blasts)
migrating cells
source: www.amnis.com
Investigation of
the phagocytosis
phagocytosis of fluorescent
latex particles by dendritic
cells – conventional flow
cytometry
Measurements of the “difficult” small cell populations
can be achieved by multicolour cytometry
e.g. identification and investigation of different monocyte cell subpopulations in the mouse peripheral blood
used antibodies:
CD45+ cells:
leukocyte marker for excluding the erythrocytes
Ly6C+ cells:
myeloid cell marker (monocytes, MF, neutrophyls)
Ly6G- cells:
Ly6G is a good neutrophil marker in the mouse
CD115+ cells:
M-CSF receptor mainly on macrophages
F4/80+ cells:
different macrophages an monocytes can express it
MHCII+ cells:
a good marker for antigen presenting cells
4-8 colour can be relatively easily measured by the recent cytometers
(You should use “fluorescence compensation controls” to consider the spectral
overlaps of the different fluorescent dyes)
Using more and more colours increase the hardness/trickiness of the experiments
Spectral cytometry
The detailed measurements of the fluorescent
spectra helps to identify different overlapping
colours in the multicolour experiments (including
mathematical methods)
by David Buschke,
Other possibility for “multicolour” cytometry
Using different type, pin sharp “spectral” labels:
heavy metal–conjugated antibodies
(mainly lantanoids)
In mass spectroscopy this labels are very easily discriminated from each other
MASS CYTOMETRY
Cell separation
Physical isolation of the cells of interest from a heterogeneous population
Differences in the physical , biological or immunological properties of the cells
are utilized to separate the cells
physical – density, size
cell biological – adherence, phagocytosis, sensitivity to the medium
immunological – antigen differences (surface!)
Differences in cell surface receptor expression are convenient possibilities for
the separation of different cell types
Characteristics of the separation:
• purity
• recovery, yield, loss
• viability of the cells
Separation
Base strategies:
positive separation – labeling and separation of the cells of interest
e.g. Labeling of a cell surface molecule (receptor!) by a fluorescent antibody.
The cells become affected both by the separation environment and the
antibodies bound to the receptors. The purity of the separation is generally
high.
negative separation – get rid of the labeled unwanted cells (depletion)
The cells become affected only by the separation environment This is the
preferred strategy in the functional examinations.
The two procedure can be used in combinations
Peripheral blood is an easily accessible source for the separation of
human immune cells and for plasma components
Separation of the plasma from the cellular components:
Separation by filtration (simple membrane or holofiber „membrane”)
Pore diameter for plasma separation: 0.2 to 0.6μm
The different density parts of the anticoagulated blood is separated to three
parts in undisturbed tube:
• bottom: sedimented red blood cells
• top: cell free plasma
• the intermediate layer is called „buffy coat” contains the leukocytes,
platelets
The process can be accelerated by centrifugation
(clinical procedures)
Apheresis (ancient greek ἀφαίρεσις) -“to take away”
Separating one particular component of the blood and
returning the remainder to the circulation
• Donor apheresis
• Therapeutic apheresis
Donor apheresis:
• Plasmapheresis – processed (e.g. IVIG) or fresh frozen plasma for immunodefficient
patients or in acute infections as passive immunization
• Plateletapheresis (trombocytapheresis) – concentrated platelets for inherited or
induced thrombocytopenia (infections, chemotherapy, irradiation) or in the case of
thrombocyte disfunction
• Red blood cells (erythrocytapheresis) - for patients with anemia (inherited or
internal/external blood loss by surgery or trauma)
• Leukapheresis – buffy coat, mainly for autotransplantation
• isolating and protecting the leukocytes before chemotherapy
• monocyte separation for dendritic cell therapy
• leukapheresis after the mobilization of bone marrow stem cells – for stem cell therapy
or bone marrow transplantation (autologous, allogeneic)
(clinical procedures)
Therapeutic apheresis:
• Removing abnormal/disfunctional components of the blood (humoral or
cellular)
• Replacing a component with a healthy donor apheresis product
• Blood component alteration (ex vivo therapy)
• Leukapheresis – Extreme high leukocyte number can lead to hemostatic disorders in
leukemia (accompanying shortness of breath, vision changes). Inflammatory cell number can be
decreased in autoimmune chronic inflammatory diseases (ulcerative colitis, rheumatoid arthritis)
• LDL apheresis – Removal of low density lipoprotein from the plasma in patients with familial
hypercholesterolemia (adsorption with ApoB affinity column or precipitation with acetate)
• Thrombocytapheresis – In essential trombocythemia/e.trombocytosis (rare disease) the
disfunctional very high thrombocyte number (with thrombosis and bleedings) can be lowered
rapidly in the rare cases of life threatening emergency situations
• Erythrocytapheresis - Removing abnormal red blood cells in patients experiencing sickle
cell crisis (in sickle-cell anemia)
• Plasmaexchange – Removing/replacement the plasma with autoimmune antibodies in
various autoimmune diseases (combined with immunosuppression) (eg. Myasthenia gravis,
Guillain-Barré syndrome, lupus, Goodpasture syndrome, Antiphospholipid antibody syndrome,
Behcet syndrome, etc….)
• Immunoadsorption with protein A/G – removal of allo- and autoantibodies (in
autoimmune diseases, transplant rejection, hemophilia) by directing plasma through protein A or
G-agarose columns
Continuous Flow Apheresis Systems
Continuous Flow Centrifugation (CFC)
Cross section representation of an old
fashioned washable apheresis centrifuge bowl
Thrombocytes and thrombocyte free plasma can
be separated by appropriate CFC methods
The blood or the “buffy coat”
cells are too “contaminated” with
erythrocytes for some purposes
Ficoll-Paque
(1.077g/ml)
Ficoll helps to separate the
near similar density cells
(from Google pictures)
(Nature Protocols http://www.nature.com/nprot/journal/v3/n6/images/nprot.2008.69-F1.jpg)
Ficoll-Paque - density based cell separation
peripheral blood
centrifugation
pipetting the cell
layer into a new tube
plasma
mononuclear
cells
(PBMC)
ficoll
Ficoll can be filled under
the blood with a thin
pipette or the blood can
be layered on the Ficoll
neutrophil
granulocytes
RBCs, dead cells
separated
peripheral
blood
mononuclear
cells
Granulocytes are also missing from the Ficoll separated
blood samples
Separation with the lysis of
the erythrocytes
Density based cell separation
(Ficoll)
missing
granulocytes
Percoll – best way to separate neutrophil granulocytes
Physical chemical characterization of Percoll. I-III. Laurent, T.C. et al.
Colloid Interface Sci. 76, 124–145 (1980).
stepwise
or
continuous
pecoll gradient
Romanian J. Biophys., Vol. 14, Nos. 1–4, P. 53–58, Bucharest, 2004
Rosette formation
Red blood cells can be coupled to other cells with the help of
antibodies
(a special type of agglutination)
The unwanted cells closed into erythrocyte rosettes
can be depleted by Ficoll separation:
Red blood cell rosettes can be used for cell separation
(depletion)
negative separation
Separating or depleting adherent cells
(both for negative and positive separation)
Simple, cheap, but only for adherent cells.
The purity is not too high
Antibody mediated separation methods
Complement mediated lysis
cell or cell type specific antibodies
complement factors
LYSIS
only for negative separation (depletion)
(Erythrocytes can be selectively lysed in a slightly hypotonic ammonium chloride buffer)
Antibody mediated separation methods
Antibody ”panning”
(both for negative and positive separation)
cell specific antibodies on the surface
magnetic cell separation
Simple magnetic separation of macrophages
Phagocytic cells engulf small iron particles. A strong magnet can
be used to separate these cells from the others.
Immuno magnetic cell separation – “MACS”
(1st step)
cell specific antibody
paramagnetic bead
MACS (2nd step)
Magnetic cell separation - MACS (3rd step)
separation of the
labelled cells
(positive separation)
MAGNET
MAGET
separation column
lots of
similarities with
the procedures of
the affinity
column
depletion or separation of the non
labelled cells (negative separation)
Magnetic separation columns
CliniMACS – closed system magnetic cell separation
CliniMACS Plus
CliniMACS® Prodigy
The paramagnetic particles are very small and
possibly don’t influence the cells’ functions
CD8+ T cells
„DETACHaBEAD”
Polyclonal antibodies against the Fab part of the "paramagnetic" antibodies.
Possibly involving anti-idiotype antibodies. They can help to detach the
"paramagnetic" antibodies from the cell surface. (competition between the cell
surface antigen and the anti-idiotype antibodies)
FACS (Fluorescence Activated Cell Sorting)
any detected cell population can be separated
• light scattering
• fluorescence
PMT 4
sample
PMT 3
PMT 2
Flow cell
PMT 1
Laser
CD antigen
cell types
function
ligand
CD3
T cells
T cell antigen receptor
signalling chain complex
CD4
helper T cells, plasmacytoid dendritic cells (pDC), monocytes
co-receptor of the T cell
antigen receptor, (also HIV
entry receptor)
MHC II, (HIV)
CD5
T cells, (B cell sub-population: B1)
cell adhesion, signalling (costimulation)
CD72
CD8
cytotoxic T cells, (NK, some  T
co-receptor of the T cell
antigen receptor
MHC I
cells)
CD14
Monocytes, macrophages,
some granulocytes
part of the LPS signalling
receptor complex
LPS, LBP
CD19
B cells
part of the B cell Ag receptor
co-receptor complex
C3d, C3b
(CD19/CR2(CD21)/CD81)
CD28
T cells
co-stimulation
(B7-1, B7-2)
CD80, CD86
CD34
hematopoietic progenitor cells,
endothelial cells
cell adhesion, …
CD62L
(L-selectin)
CD56
NK cells, (some T and B cell sub-
homoadhesion (N-CAM
isophorm)
population)
CD80, CD86
(B7-1, -2)
professional APC: DC, B,
monocytes, macrophages
co-stimulation, cell adhesion
CD28, CD152
examples:
•B1 cell separation: CD19+ & CD5+
•NKT cell separation: CD3+ & CD56+
NKT cells
NK cells
lymphocytes
Multiple step magnetic separations are needed to achieve similar result:
Can you figure it out?
FACS:
The constant vibration of the flow cell produces drops from
the flow stream in a stable position
breakoff point
vibration
+
+
+
+
+
+
+
+
+
Laser
electrically charged
deflection plates
+
+
+
+
When the targeted cell reach
the breakoff point, a short
charge is applied on the
stream, and the detaching
drop get charged.
+
+ +
+ +
-
electrically charged
deflection plates
--- collecting tube
collecting tube
The control panels of a FACS sorter