citometria de fluxo com material obtido em parte da: purdue university cytometry laboratories (
TRANSCRIPT
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Citometria de fluxo
Com material obtido em parte da:
Purdue University Cytometry Laboratories(http://www.cyto.purdue.edu/flowcyt/educate/pptslide.htm)
Agradecimentos:•Dr. Robert Murphy, Carnegie Mellon University, Pittsburgh, PA
•Dr. J. Paul Robinson, Purdue University Cytometry Laboratories
Dr. Carleton Stewart,RPCI Laboratory of Flow Cytometry, Buffalo,NY
•B. H. Davis, William Beaumont Hospital, Royal Oak, Michigan
Faculdade de Medicina da
Universidade Federal da Bahia
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O que é a citometria de fluxo:
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Avaliação de sub-populações celulares: quadrantes CD4 e CD8.
Log FITC Fluorescence (CD8).1 1 10 100 1000
Lo
g P
E F
luo
resc
ence
(C
D4)
.1
1
10
10
01 2
3 4
45% 2%
26%27%
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Avaliação de ativação celular: explosão respiratória.
DCFH-DA
DCFH-DADCFH-DA
DCFHDCFH
DCFDCF
H OH O 2 22 2
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Análise de divisão celular.
GG22
MM GG00
GG11
ss
0 200 400 600 800 1000
GG00GG11
ss GG22 MM
DNA AnalysisDNA Analysis
DNA contentDNA content
Count
2N2N 4N4N
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Análise de DNA
0 200 400 600 800 1000
PI Fluorescence
Cou
nts
0 7
5 1
50 2
25 3
00
2N2N 4N4N
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Influxo de cálcio
Time (Seconds)0 36 72 108 144 180
RA
TIO
[sho
rt/lo
ng]
0 2
00 4
00 6
00 8
0010
00
StimulationStimulation
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Análise de reticulócitos
log Thiazole Orange.1 1000 100 10 1
Count
0
15
0
11
2
75
3
7
RMI = 0RMI = 0
log Thiazole Orange.1 1000 100 10 1
Count
0
15
0
11
2
75
3
7
RR11RR22RR33RR44
RMI = 34RMI = 34
RR11RR22RR33RR44
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Citômetro de fluxo
Computer SystemDetector &Mechanical Fluidics
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Célula de Fluxo
Injector Tip
FluorescenceFluorescencesignalssignals
Focused laserFocused laserbeambeam
Sheath fluid
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90 Degree Light Scatter
FALS Sensor
90LS Sensor
Laser
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Detectores de fluorescência
Fre
q
Fluorescence
FALS Sensor
Fluorescence detector(PMT3, PMT4 etc.)
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Marcadores fluorescentes
Fluorescein (FITC)
400 nm 600 nm 700 nm
Relat
ive In
tensit
y
Wavelength
500 nm
ExcitationEmission
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Cromógenos
EthidiumEthidium
PEPE
cis-Parinaric acidcis-Parinaric acid
Texas RedTexas Red
PE-TR Conj.PE-TR Conj.
PIPI
FITCFITC
600 nm300 nm 500 nm 700 nm400 nm
457350 514 610 632488Common Laser Lines
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Imunofenotipagem: marcação celular
CD2CD2CD4CD4
CD # = cluster designation number
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Imunofenotipagem
Log FITC
1000 100 10 1
11
0
.1
Cou
nt
3
0 7
00
1
50
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Light scatter gating
90 Degree Scatter0 200 400 600 800 1000
Forw
ard
Sca
tter
0 2
00 4
00 6
00 8
0010
00
8
15
20
30
40
50
100
200
1000
Lymphocytes
Monocytes
Neutrophils
Side Scatter Projection
Fo
rwa
rd S
catte
r P
roje
ctio
n
Scale
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Avaliação de sub-populações celulares: quadrantes CD4 e CD8.
Log FITC Fluorescence (CD8).1 1 10 100 1000
Lo
g P
E F
luo
resc
ence
(C
D4)
.1
1
10
10
01 2
3 4
45% 2%
26%27%