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Measuring the functional activity of T and B lymphocytes
Polyclonal activation of T and B cellslectin-induced activationα-IgM, α-CD3 or α-TCR antibodyallogeneic T cell activation(examination of the immediate-early activation events)
T and B cell responseactivation markersproliferative response: 3H-thymidine incorportion
CFSE fluorescence decreasecell cycle events
Antibody or cytokine production (ELISA, bioassay, CBA)
Determinating the number of activated T and B cells after the administration of the antigen
ELISPOT, Intracellular cytokine stainingMHC tetramers
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Phases of the humoral immune response(review)
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Phases of T cell response(review)
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Lymphocyte function can be investigated by polyclonal T/B-lymphocyte activator materials
Immunodeficiencies mainly characterized by different functional immunoassays
Lymphocyte activation by specific antigen is hardly detected, because of the low number of the antigen specific cells
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Polyclonal activation of lymphocytes by LPS, lectins, PMA/ionomycin
A B C
BCR or TCR-specifc antibodies could activate the lymphocytes also
(PMA activates protein kinase C)
TLR4
B cell T cell T cell
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Polyclonal B cell activators
In the presence of cytokinesyesAnti-Ig
yesyesEBV (transforming effect)
yesnoSpA (superantigen, staphylococcus protein A)
yesnoPWM (pokeweed mitogen)
Human B cells
Ig secretionT cell dependencyActivator
Polyclonal T cell activatorsPhytohaemagglutinin (PHA) lectin Canavalia ensiformis
Concanavalin A (ConA) lectin Phaseolus vulgaris
anti-CD3 Monoclonal antibody
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Pokeweed (PWM)(Phytolacca americana) – formerly used for coloring red wine(toxic: triterpene saponin)ChenopodialesPhytolaccaceae
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Phytohaemagglutinin (PHA) Canavalia ensiformis – Jackbean, Sword bean
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Concanavalin A (ConA) Phaseolus vulgaris – bean
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EXAMINATION OF T AND B CELL FUNCTIONS
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Receptor crosslinking(immediate)
phosphorylation steps(seconds-minutes)
- Western blot- Bead array
I.c Ca2+ increase - FACS, microscopy
Gene activation - RT-PCR
Cytokine synthesis
Cytokine secretion
- i.c cytometry
- ELISA, ELISPOT- Bioassay
Antigen receptors (TCR, BCR), and different other receptors (eg. cytokine receptors)
Cell-cycle/apoptosis - DNA content- IN antigens
Cell division - 3H-thymidine, CFSE, MTTLymphocyte activationThe examination often requires specific
Ag-Ab reactions
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Western blottingSteps:
1. sample preparation (cells, tissues)2. gel electrophoresis3. blotting4. labeling5. development
use:
Identification of defined components from protein mixtures by antigen specific antibodies
Anode(+)
Cathode(-)
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• SDS-PAGE gel resolved into single protein bands (overlap possible)• Presence of a protein is determined by hybridizing the proteins, transferred or applied to a membrane, with the relevant antibody
StandardProtein sample
SDS-PAGE Membrane Western blot
Antibody recognizes epitope in specific protein
Western Blot
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Western Blot
• Used to detect specific proteins in a sample
• Proteins separated by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), transferred to a membrane
• Primary (1st) antibody (monoclonal or polyclonal) used to detect protein
• Enzyme linked 2nd antibody (e.g. horseradish peroxidase-linked) used to detect 1st antibody
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Investigation of the presence or absence ofInvestigation of the presence or absence ofBrutonBruton’s’s t tyyrorosine sine kinkinasease (BTK) (BTK) by by Western blotWestern blot
X-linked agammaglobulinemia. XLA patients do not generate mature B cells, which manifests as an almost complete lack of antibodies in their bloodstream.
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Futatani T et al. Blood 1998;91:595-602
Investigation of the presence or absence ofInvestigation of the presence or absence ofBrutonBruton’s’s t tyyrorosine sine kinkinasease (BTK) (BTK) by flow cytometryby flow cytometry
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Fluo-3 AM – excitable by blue light Indo-1 AM – excitable by UV light
These indicator dyes bound to apolar groups (e.g. acetoxy-methylester: AM) cross the cell membrane, in the cell, esterases cleve them so the fluorochromes become polar and are
trapped in the cell
An increase in cytoplasmic Ca2+ levels can be detected by
fluorescent indicator dyes./Fluo-3 or Indo-1/
Detection of intracellular Ca2+ concentration
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Measurement of Ca2+ signal by flow cytometry
Flu
ore
scen
ce p
rop
ort
ion
al
wit
h I
ntr
acel
lula
r C
a2+ l
evel
time
basic signal
activation of cells
e.g. Fluo-3or
Indo-1
You can detect by fluorimeter also
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Measurement of Ca2+ signal by flow cytometryT cell hybridoma specific for influenza virus hemagglutinin protein-derived
peptide - Ca2+ signal by antigen presentation
non-activated T cells
T cell activation(APC - T cell)
activated T cells
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Immunohistochemistry
Labeled antibodies added to fixed tissue sections detect the distribution of the chosen antigen within the tissue or within the cells of a particular tissue
• Immunofluorescence•Fluorescent dye coupled to antibody
FITC – fluorescein isothiocyanate (green)PE – phycoerythrin (orange)
• Immunoenzyme method• enzyme-coupled antibody
P – peroxidase PA – alkaline phosphatase(Substrates converted into an insoluble compound)
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Tissue sample
Fixation
Section before staining
Freezing
Sectioning
Immunohistochemistry
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Immunohistochemistry ABC Method
Secondary antibody
Avidin
Cells
Slide
Primary antibody
Biotin
Enzim
X
Tissue sample
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Classical histochemistry
Acute bronchopneumonia (hematoxilin- eosin staining)
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Immunohistochemistry (CD68+ macrophages and lymphocytes, granuloma)
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Antinuclear autoantiboies (ANA) from the serum of a SLE patient can be visualized in cell culture (Hep-2) by indirect fluorescent labeling (immunofluorescence)
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Immunohistochemistry using fluorescent detectionDetection of actin microfilaments
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A fixed and permeabilized skin fibroblast.
Mitochondria were labeled with mouse IgG (anti–OxPhos Complex V) and visualized using goat anti–mouse IgG conjugated with orange-fluorescent Alexa Fluor 555.
F-actin was labeled with green-fluorescent Alexa Fluor 488 phalloidin (a mushroom toxin).
Nucleus was stained with TO-PRO-3 iodide.
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Peroxisome labeling in fixed and permeabilized pulmonary artery endothelial cell.
Peroxisomes were labeled using an antibody directed at peroxisomal membrane protein 70 and detected with Alexa Fluor 488–labeled goat anti–mouse IgG.
Mitochondria were stained with MitoTracker Red prior to fixation;
Nuclei were stained with blue-fluorescent DAPI.
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Flow cytometryFlow cytometry
An immunofluorescent method that mutually complements the fluorescent microscopy
• Investigation of different cells or particles travelling high velocity in flow
• Detects fluorescence intensity and scattered light of the labeled cells
• Can investigate enormous number of cells in short period Can investigate enormous number of cells in short period of timeof time
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Why flow cytometry
Most cells in the immune system can be found in free or loosely adherent form. They can be easily suspensed and labeled by fluorescent antigen specific antibodies, and then they can be examined cell by cell.
The cells’ light scatter and immunofluorescent properties can be analyzed statistically (eg. percentages of different cell populations)
Rare cell populations can be identified and examined (eg. 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. (eg. cell activation, disease progression)
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Benchtop flow cytometer
Sorter - flow cytometer(FACS station)
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Example Chanel Layout for Laser-based Flow CytometryExample Chanel Layout for Laser-based Flow Cytometry
PMT 1
PMT 2
PMT 4
The emited fluorescent light can be separeted to components by
special mirrors and filters
Laser
PMT 3cell
suspension in tube
photodetectors
flow cell
forward light scatter detector
(PMT=photo-multiplayer tube)
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Laser
Light scatter and fluorescence
Forward angle light scatter
sensor(FSC, FALS)
Side light scatter(SSC) and fluorescence detectors
Can be loosely considered as a
representation of the particle size
represents the granularity of the cells
Multocolor staining can be used to identify cell sub-populations
* autofluorescence – presence of piridins and flavins.
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BNKTh Tc
Example:
Measurement of CD4+ (helper) and CD8+ (cytotoxic) T cell ratio (eg. monitoring AIDS progression)
Labeling:
FITC labeled anti-CD4 antibody(α-CD4-FITC)PE labeled anti-CD8 antibody (α-CD8-PE)
Lymphocytes in the periferial blood
Fluorescent microscopy
Immunophenotyping
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focu
sed
lase
r bea
m
high velocity flow stream(in cuvette or stream in air)
CD4FITC
CD
8P
E
de
tec
tor
detecting CD4-FITC labeled (TH) cell
signal processing unit
screen
a dot representing aCD4+ CD8- cell
increasing light intensity
microscopy:
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de
tec
tor
detecting the PE labeled cell(CD8-PE)
CD
8P
ECD4FITC
signal processing unit
increasing light intensity
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de
tec
tor
detecting the unlabeled cell(eg.B cell) by autofluorescence
CD
8P
ECD4FITC
Signal processing unit
increasing light intensity
microscopy: dim (autofluorescent)
cell
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quadrantstatistics
CD
8P
ECD4FITC
38%
0%
44%
18%
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Graphical representations 1.
dot-plot
contour-plot
density-plot
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Graphical representations 2.
Histogramm
homogenous cell population is normally distributed (Gaussian)
Numeral intensity values:
~ 7 ~ 1300
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Different cell types - Different cell types - characteristic light scatteringcharacteristic light scattering
forward light scattering (FSC)
(„size”)
side light scattering (SSC)(e.g. granulated)
granulocytes
monocytes
lymphocytes
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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)
Only the major cell types can be identified
1 Noise2 Nucleated Red Blood Cells3 Platelet Clumps4 Lymphocytes and Basophils5 Large Unstained Cells6 Monocytes7 Neutrophils8 Eosinophils
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Characterisation of immune cells using cell Characterisation of immune cells using cell surface markerssurface 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+ = 1.6Normal CD4+ T cell count = 600 – 1400/l
AIDS = CD4+ T cell count <200/l
- increase of CD5+ B cells – typical for some B cell Leukemias
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WAS: Wiscott-Aldrich Syndrome
XLA: X-linked Agammaglobulinemia
Inhibited B cell development: Lack of CD19+ B cells
A typical symptom: Lacking or decreased CD43 expression
Diagnosis of immunodeficiency by flow cytometry
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Intracellular cytokine detection by immunofluorescence
cytokine specific antibody with fluorescent labelling
- the cell membrane should be permeabilized (detergent)
- the cells should be fixed previously avoiding the decomposition of the cells (e.g. aldehyde fixation)
cytokines
- optionally the cells could be labelled by some cell type specific
antibody in the beginning (e.g. CD4)
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One can determine which cell type produced the cytokines!
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ELISA
ELISA plate
Enzyme Linked Immune Sorbent Assay
well
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enzyme linked immune sorbent
Antibody conjugated with enzyme
enzyme
Antigen/antibody adsorbed to solid surface
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Enzyme activity in ELISA is directly proportional to the amount of antigen
present
Enzyme activity is measured by the color reaction due to conversion of substrate
Similar principle applies to many other antibody-based detection methods
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Basic setups in ELISA, immunohistochemistry, flow cytometry
Direct method Indirect method
Antigen
Primary antibodies
Label
Label
Secondary antibodies
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For antigens present at low concentration in complex biological samples
Removal of unbound material
Blocking free plastic surface with inert
protein
Removal of unbound protein
Addition of biotinylated antibody specific to a different epitope on
target protein
Removal of unbound material
Addition of avidin-conjugated enzyme
Addition of substrate
Coating with Ag-specific „capture”
antibody
Addition of antigen- containing solution
Removal of excess enzyme
Steps of combined sandwich ELISA
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May be you have already met such kind diagnostic tools, or you are going to meet them during your career
Eg. detection of human chorionic gonadotropin in serum or urine
(pregnancy test)
The principles of these tools are similar as the ELISA assay you have met before.
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hCG Rapid One-Step Immunochromatographic Assay strip
nitrocellulose membrane(signal detection pad)
glass fiber membrane with visually labeled detection antibodies
front view side view
hCG capture antibody lane
control antibody lane(detection antibody capture)
absorbtion pad (cellulose)
sample application pad
urine
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hCG capture antibody line
control antibody line
detection antibodies
hCG
control line (C)
test line (T)
hCG + hCG negative
detection antibody capture antibodies
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hCG line( bound hCG)
control linedetection antibody
capture antibodycontrol line
test line
hCG positive hCG negative
Competitive system
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ELISA plates - resultsELISA plates - results
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ELISPOTEnzyme Linked Immuno-Spot
The principles are similar to ELISACapable to determine the number of cells that produce Ig, cytokines, chemokines, granzymes and other soluble effector moleculesThe sensitivity allows the determination of 1 activated cell among 300,000 others, so it can reveal activated effector cells not only after polyclonal but after antigen specific activationThe first steps should be done in aseptic conditions
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ELISPOT- coating with antigen specific capture antibodies
- blocking
- administration of the cells
- administration of biotin conjugated antigen specific secondary antibody
- avidin-enzyme conjugate
- administration of the unsoluble chromogenic substrate (AEC 3-amino-9-
ethylcarbazol)
(activation, incubation)
- washing
A spot showing the place of the cytokine
producer cell
Upper view of a well on an ELISPOT plate with the generated spots
The process
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It can be evaluated by microscopy (slow, manual process) or you can use “ELISPOT plate reader” (fast + standardizable spot number and size determination)
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Very sensitive cytokine determination can be achieved by cytokine sensitive or cytokine dependent cell lines. The presence or absence of cytokines determines the fate of the indicator cells that could be
cell proliferation or cell death.
BioassayBioassay
Wehi 164 cell line
CTLL2 cell line
IL-2 is present no IL-2
TNF is present no TNF
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Bioassays could be equivocal, because of the cytokine cross reactivity of indicator cells: eg. the IL-2 dependent CTLL2 cells can proliferate in the presence of high IL4 concentration also.
Living cells can be visualized by colorimetric assay (eg. MTT), or their proliferation can be measured by other methods.
cytokine concentration
IL-2
TNF
CTLL2CTLL2
Wehi 164Wehi 164
MTT assay living cells convert the stain to purple-blue
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Cytokine arrayCytokine array(The process is similar to the procedures of Western-blot after the blotting step)
( - )
IFN
( + ) IL-2 IL-4 IL-5
…
IL-12 …
multiple antigen specific antibodies bound to membrane
unknown cytokine containing solution
„Luminescent antibody” mixture
Multiple cytokines can be detected rapidly in the same procedure
Disadvantage – defined volume sample needed to cover the surface of the membrane
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cytokine production ofmoDC activated by CD40L andCD40L+SLAM combination
Réthi és mtsi. 2006
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method: RT-PCR, QRT-PCR
Investigation of gene activationInvestigation of gene activation
Activation of T cells can be monitored by the detection of the transcribed mRNA of the activated genes.
e.g. activation of cytokine genes
cells RNA isolation
RNA (reverse transcriptase) cDNA
cDNA (PCR) determination of the length and quantity
RT-PCR: agarose gel (densitometry)QRT-PCR: fluorescent method(TaqMan probe (FRET) or dsNA intercalating fluorochrome SYBR green)
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PCR
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Characteristics of the separation:
• purity• recovery, yield, lost• viability of the cells
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
(differences in cell surface receptor expression is often available – there is a possibility to further investigate the separated living cells )
physical – density, size cell biological – adherence, phagocytosis, sensitivity to the
medium immunological – antigen differences (surface!)
Cell separationCell separation
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Base strategies:
positive separation – labeling and separation of the cells of interest
eg. 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.
SeparationSeparation
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The different density parts of the anticoagulated blood has been separating to three parts in undisturbed tube:bottom: sedimented red blood cellstop: cell free plasmathe intermediate layer is called „buffy coat” contains the leukocytes, platelets
The process can be accelerated by centrifugation
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(Nature Protocols http://www.nature.com/nprot/journal/v3/n6/images/nprot.2008.69-F1.jpg)
(from Google pictures)
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Ficoll-Paque: density based cell separationFicoll-Paque: density based cell separation
peripheral blood
pipetting cells on ficoll
centrifugation
separated cells
plasma
ficoll
Red blood cells
mononuclear cells
(PBMC)
neutrophilgranulocytes
pipettig the „ring” containing the
mononuclear cells to a new tube
to get rid of ficoll
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Magnetic immunoseparation (MACS)
paramagneticbead
antigen specific antibody
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MACS
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Magnetic cell separation (MACS)
MA
GN
ETM
AG
NE
T
column
depleting or selecting unlabeled cells
(negative separation)
separation of labeled cells(positive separation)
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Magnetic column