bd facscalibur™ flow cytometer facs (fluorescence ... · lymphocytes (r1) 1,11% 0,05% 69,27%...
TRANSCRIPT
![Page 1: BD FACSCalibur™ flow cytometer FACS (Fluorescence ... · Lymphocytes (R1) 1,11% 0,05% 69,27% 29,56 % Perforin FITC IFN-gamma PE CD3+ CD8+ Lymphocytes (R1 and R2) Simultaneous phenotypic](https://reader031.vdocument.in/reader031/viewer/2022022115/5c72f57409d3f2601f8c845a/html5/thumbnails/1.jpg)
BD FACSCalibur™ flow cytometer
FACS (Fluorescence Activated Cell Sorting)
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Un citometro a flusso è essenzialmente costituito di:
• Sistema fluidico per il trasporto del campione e la sua focalizzazione idrodinamica
• Sistema ottico • Circuito di rilevazione • Elettronica per l’acquisizione, elaborazione e
presentazione dei dati.
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Sample
Sheat pressure regulator
waste Sheat fluid
Air pump
Flow chamber
Air filter
Sample pressure regulator
Filter
The fluidic system
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Hydrodynamic focusing
High sample flow rate 60µl/min
Laminar Flow
Laminar Flow
Sheath Sheath Sheath Sheath Sample Sample
Low pressure High pressure
Sample core
Low sample flow rate 12µl/min
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LIGHT SCATTER
FSC
SSC
Incident light
source
Diffrazione Proporzionale all’area della cellula. Rilevata lungo l’asse della luce incidente 0°.
Rifrazione e Riflessione proporzionale alla granularità cellulare e alla complessità. Rilevate a 90° rispetto la luce incidente
LASER
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Discrimination of different cell populations in human blood based on light scattering
Sid
e S
catte
r Forward Scatter
lymphocytes
granulocytes
monocytes
Lysed whole blood
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Characterization of unstained cells using light scatter
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The fluorescence phenomenon
E’ il fenomeno per cui una molecola colpita da una radiazione luminosa di definita lunghezza d’onda (λ) ne emette un’altra a λ maggiore e ad energia inferiore.
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FLUOROCROMI
Molecole fluorescenti che assorbono energia dal laser e rilasciano l’energia assorbita per:
- Vibrazione e dissipazione del calore - Emissione di fotoni di una lunghezza d’onda più lunga. Sono coniugate a ligandi o anticorpi monoclonali specifici per
molecole biologiche con significato antigenico o recettoriale posizionate sulla membrana cellulare, nel nucleo o nel citosol; altri “colorano” direttamente altre sostanze (DNA, RNA, proteine, ioni citoplasmatici).
Anche le cellule non marcate con alcun fluorocromo presentano una debole fluorescenza di fondo, l’autofluorescenza, misurabile.
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Absorption and emission spectra of a fluorochrome (Stokes shift)
Ogni fluorocromo presenta una caratteristica banda di λ per l’eccitazione e per l’emissione
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I fluorocromi attualmente più usati in citofluorimetria sono:
• FITC • PE • Fluorocromi tandem • APC • PI (Propidium Iodide) • PerCP
Ciascun fluorocromo è caratterizzato da una particolare BRILLANTEZZA.
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Absorption and emission spectra of different fluorochromes
RPE APC FITC PerCP PI
400 450 500 550 600 650 700
1000
800
600
400
200
0
400 450 500 550 600 650 700
RPE APC FITC PerCP PI
Wavelenght (nm)
Absorption spectra Emission spectra
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Tandem fluorochromes
767 650 APC-Cy7 (PharRed)
670 488 PE-Cy5 (Cy-chrome)
695 488 PerCP-Cy5.5
Composti fluorescenti costruiti unendo fra loro molecole con proprietà fotofisiche complementari, atte a sfruttare il principio di trasferimento dell’energia.
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FITC FITC
FITC
FITC
FITC
FITC
FITC
FITC
FITC
FITC
Fluorescence intensity
Num
ber o
f eve
nts
Emitted fluorescence intensity ∝ binding sites
Correlation between number of binding sites and fluorescence intensity
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Staining of cells with fluorochrome conjugated antibobies specific for surface antigens
Antigen A
Antigen B
anti-B Antibody
anti-A Antibody
fluorochromes
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washing Incubation
acquisition
Incubation washing
acquisition
Single and three colours staining
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Acquisition and
Analysis
Incubation
washing
Second antibody Goat Anti-Mouse Ig FITC
First unconjugated mouse antibody
Incubation
washing
Indirect staining
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Optic system of a flow cytometer
FITC
SSC
PE / PI
PerCP 7-AAD PE-Cy5 FSC
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460 500 540 460 500 540 460 500 540
SP 500 LP 500 BP500/50
Longpass Shortpass Bandpass
Optical filters
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FL1
SSC
FL3
FL4
670LP
661/16
585/42
488/10
90/10 Beam Splitter
DM 560SP
Fluorescence Collection Lens
DM 640LP
Half Mirror
488/10
. FSC Diode
Focusing Lens
FL2
530/30
Beam Combiner Flow Cell
Flow Cell
Optic system of a BD FACSCalibur flow cytometer, equipped with two lasers
I segnali di fluorescenza emessi dalla cellula vengono raccolti da lenti poste ortogonalmente al fascio di eccitazione, selezionati con opportuni specchi separatori di fascio, specchi dicroici e filtri ottici e portati ciascuno ad un diverso PMT.
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FLUORESCENCE MEASUREMENTS
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Generation of an electrical pulse
Time
Volta
ge
Time
Volta
ge
Time Vo
ltage
Sensori denominati PMT o detectors trasformano i segnali ottici in intensità di corrente elettrica, oltre ad amplificare lo stesso segnale in maniera lineare o logaritmica.
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Conversion of electrical pulses in digital values
Time (µSec)
Volts
Pulse area
Puls
e he
ight
pulse
width
40 0
1000
Channels
Analog to digital
converter 0
FSC
SS
C
FL1
FL2
FL3
I segnali provenienti dai PMT sono impulsi analogici, ossia proporzionali in maniera continua alle dimensioni del parametro misurato. Un convertitore “spezzetta” i valori continui rendendoli discreti a seconda del numero di canali a disposizione.
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1000 500 0
Num
ero
di C
ells
510 500 490
Incremento di intensità
Histogram plot
Il diagramma ad istogrammi rappresenta graficamente la misura di un singolo parametro. Gli eventi che si accumulano nei vari canali vanno a costruire un diagramma di distribuzione.
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Histogram analysis
La statistica si basa sulla impostazione di cursori che definiscono le aree di interesse calcolando gli eventi che cadono al loro interno.
101 FL-1
even
ts
even
ts
101 FL-1
even
ts
even
ts
Unstained sample Stained sample
marker
Background fluorescence
Antibody + cells
Antibody - cells
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HISTOGRAM
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Dot plot
400 800 1000 0 0 200 400 600 800 1000 0
200
400
600
800
1000
FL1
FL2
840
85
245 638 FL1
FL2
0
200
400
600
800
1000
200 600
Il diagramma di dispersione a punti o dot plot rappresenta la correlazione fra due parametri, in cui ogni punto rappresenta un singolo evento dotato di un particolare valore per ciascuno dei due parametri.
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Dot plot analysis
UL
LR LL
UR
CD3 FITC
CD4
PE
UL CD3-/CD4+
UR CD3+/CD4+
LL CD3-/CD4-
LR CD3+/CD4-
Upper Left Single positive
Upper Right Double positive
Low Left Double negative
Low Right Single positive
quadrants
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DOT PLOT
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Ficoll
stratification
blood centrifuge
erytrocytes +
granulocytes
Plasma +
platelets
PBMC
Peripheral Blood Mononuclear Cells = PBMC
Ficoll
Isolation of PBMC by ficoll gradient centrifugation
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Immunophenotyping technique for determining % of CD4 and CD8 T cells
CD45 PerCp
SSC
CD3 FITC
CD
4 A
PC
CD
8 PE
CD3 FITC
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SPT-specimen processing
Vortex Tube with beads
Acquisition and Analysis
50 µL whole blood (EDTA)
20 µL Antibody mix
450 µL FACS Lysing buffer (1x)
Vortex
15 Minutes @ Room Temperature, dark
15 Minutes @ Room Temperature, dark
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SPT-sample analysis
No. of events in the bright CD45 region
No. of events in the microfluorosphere region
Total no. of microfluorospheres added
Volume of blood added
X = Absolute no. of CD4
Microfluorosphere
CD3 FITC
CD
4 P
E
CD45 PerCP
Granulocytes
Lymphocytes
Monocytes
Microfluorosphere
Sid
e S
catte
r
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ELISpot
Antigens
PBMC
MoAbAnti-IFN-γ
PBMC
Antigen
IFN-γ
Enzymatic reaction
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Capture anti-cytokine
antibody
CYTOKINE
Detection anti-cytokine antibody
Chromogen Substrate
insoluble coulored product
ELISpot
Enzyme conjugated to the detection antibody
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ELISpot
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Simultaneous phenotypic (naive/memory phenotype) and functional (IFN-γ production) characterization of antigen stimulated CD8 T lymphocytes
Naive CD45RA+CCR7+
Memory CD45RA-CCR7+
Pre-Effector CD45RA-CCR7-
Effector CTL CD45RA+CCR7-
CC
R7
CD45RA
CC
R7
CD45RA
IFN
-gam
ma
CD8
R1
R2
IFNγ-positive cells (R1) IFNγ-negative cells (R2)
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Forward Scatter
Side
Sca
tter
CD3 APC
CD
8 Pe
rCP
Lymphocytes (R1)
1,11% 0,05% 69,27% 29,56 %
Perforin FITC
IFN
-gam
ma
PE
CD3+ CD8+ Lymphocytes (R1 and R2)
Simultaneous phenotypic and functional (IFN-γ production and cytotoxic potential) characterization of antigen stimulated T lymphocytes
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Class I MHC Tetramer
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Analysis of proliferation by CFSE labelling
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CFSE PHA 1 µg/ml CFSE PHA 0,5 µg/ml
CFSE Not stimulated
No CFSE
Analysis of the proliferation of PHA stimulated CD8 T lymphocytes by CFSE labelling
![Page 43: BD FACSCalibur™ flow cytometer FACS (Fluorescence ... · Lymphocytes (R1) 1,11% 0,05% 69,27% 29,56 % Perforin FITC IFN-gamma PE CD3+ CD8+ Lymphocytes (R1 and R2) Simultaneous phenotypic](https://reader031.vdocument.in/reader031/viewer/2022022115/5c72f57409d3f2601f8c845a/html5/thumbnails/43.jpg)
CD
4
CFSE (Fluorescence intensity FL1)
Proliferation (cell division)
Not stimulated Stimulated with PHA
Analysis of the proliferation of PHA stimulated CD4 T lymphocytes
![Page 44: BD FACSCalibur™ flow cytometer FACS (Fluorescence ... · Lymphocytes (R1) 1,11% 0,05% 69,27% 29,56 % Perforin FITC IFN-gamma PE CD3+ CD8+ Lymphocytes (R1 and R2) Simultaneous phenotypic](https://reader031.vdocument.in/reader031/viewer/2022022115/5c72f57409d3f2601f8c845a/html5/thumbnails/44.jpg)
A B
Analisi del ciclo cellulare
![Page 45: BD FACSCalibur™ flow cytometer FACS (Fluorescence ... · Lymphocytes (R1) 1,11% 0,05% 69,27% 29,56 % Perforin FITC IFN-gamma PE CD3+ CD8+ Lymphocytes (R1 and R2) Simultaneous phenotypic](https://reader031.vdocument.in/reader031/viewer/2022022115/5c72f57409d3f2601f8c845a/html5/thumbnails/45.jpg)
Analisi del ciclo cellulare
![Page 46: BD FACSCalibur™ flow cytometer FACS (Fluorescence ... · Lymphocytes (R1) 1,11% 0,05% 69,27% 29,56 % Perforin FITC IFN-gamma PE CD3+ CD8+ Lymphocytes (R1 and R2) Simultaneous phenotypic](https://reader031.vdocument.in/reader031/viewer/2022022115/5c72f57409d3f2601f8c845a/html5/thumbnails/46.jpg)
Analisi dell’apoptosi (Ioduro di Propidio)
![Page 47: BD FACSCalibur™ flow cytometer FACS (Fluorescence ... · Lymphocytes (R1) 1,11% 0,05% 69,27% 29,56 % Perforin FITC IFN-gamma PE CD3+ CD8+ Lymphocytes (R1 and R2) Simultaneous phenotypic](https://reader031.vdocument.in/reader031/viewer/2022022115/5c72f57409d3f2601f8c845a/html5/thumbnails/47.jpg)
Analisi dell’apoptosi (Ioduro di Propidio + Annessina V)
![Page 48: BD FACSCalibur™ flow cytometer FACS (Fluorescence ... · Lymphocytes (R1) 1,11% 0,05% 69,27% 29,56 % Perforin FITC IFN-gamma PE CD3+ CD8+ Lymphocytes (R1 and R2) Simultaneous phenotypic](https://reader031.vdocument.in/reader031/viewer/2022022115/5c72f57409d3f2601f8c845a/html5/thumbnails/48.jpg)
Analisi dell’apoptosi (Ioduro di Propidio + Annessina V)
![Page 49: BD FACSCalibur™ flow cytometer FACS (Fluorescence ... · Lymphocytes (R1) 1,11% 0,05% 69,27% 29,56 % Perforin FITC IFN-gamma PE CD3+ CD8+ Lymphocytes (R1 and R2) Simultaneous phenotypic](https://reader031.vdocument.in/reader031/viewer/2022022115/5c72f57409d3f2601f8c845a/html5/thumbnails/49.jpg)
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