7/27/2019 Danijela Blogs Sept27throughOctober11
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Danijela BlogsSeptember 24-October1
7/27/2019 Danijela Blogs Sept27throughOctober11
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Macaque Storm Intro
•
Received FRTs, Recta and Lymph Nodes of over 20 macaqueTulane
• Macaques were challenged Vaginally and Rectally with SIV Gexpressing either mCherry:paGFP:Luciferase or BFP:paGFP:L
• We are blinded regarding the VPX status of these vectors
7/27/2019 Danijela Blogs Sept27throughOctober11
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BFP Expressing CellsGFP PhotoactivationSpectral Imaging
7/27/2019 Danijela Blogs Sept27throughOctober11
http://slidepdf.com/reader/full/danijela-blogs-sept27throughoctober11 4/47
BFP
Draq5 Nuclear Stain
Looking for BFP Expressing Cells
CE39_Vagina_1D
7/27/2019 Danijela Blogs Sept27throughOctober11
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BFP
Draq5 Nuclear Stain
Looking for BFP Expressing Cells
CE39_Vagina_1D
7/27/2019 Danijela Blogs Sept27throughOctober11
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BFP
Draq5 Nuclear Stain
Looking for BFP Expressing Cells
CE39_Vagina_1D
7/27/2019 Danijela Blogs Sept27throughOctober11
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BFP
Draq5 Nuclear Stain
Volume View: Click to Play
7/27/2019 Danijela Blogs Sept27throughOctober11
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BFP
Draq5 Nuclear Stain
Looking for BFP Expressing Cells
CE39_Vagina_1D
BFP
7/27/2019 Danijela Blogs Sept27throughOctober11
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BFP
Draq5 Nuclear Stain
Volume View: Click to Play
BFP
7/27/2019 Danijela Blogs Sept27throughOctober11
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BFP
Draq5 Nuclear Stain
Looking for BFP Expressing Cells
CE39_Vagina_1D
BFP
7/27/2019 Danijela Blogs Sept27throughOctober11
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BFP
Draq5 Nuclear Stain
Volume View: Click to Play
BFP
7/27/2019 Danijela Blogs Sept27throughOctober11
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BFP
Draq5 Nuclear Stain
Can we photoactivate GFP in BFP expressing cells?
CE39_Vagina_1D
7/27/2019 Danijela Blogs Sept27throughOctober11
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166 pre PA
188 post PA
12% increase
Background
prePA line
postPA line
BFP
7/27/2019 Danijela Blogs Sept27throughOctober11
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BFP
Draq5 Nuclear Stain
Can we photoactivate GFP in BFP expressing cells?
CE39_Vagina_1D
7/27/2019 Danijela Blogs Sept27throughOctober11
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136 pre PA
171 post PA
21% increase
Background
prePA line
postPA line
124 pre PA
238 post PA
48% increaseCE39_Vagina_1D
BFP
7/27/2019 Danijela Blogs Sept27throughOctober11
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BFP
Draq5 Nuclear Stain
Can we photoactivate GFP in BFP expressing cells?
CE39_Vagina_1D
7/27/2019 Danijela Blogs Sept27throughOctober11
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113 pre PA
209 post PA
46% increase
Background
prePA line
postPA line
Spectral imaging
7/27/2019 Danijela Blogs Sept27throughOctober11
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Spectral imaging
BFP Spectral Profile
Background
Expected Peak
Emission for BFP isat 457nm, so we are
right on target!
BFP
Draq5
Merge
Is BFP Emission Maxima where it is Expected?
CE39_Vagina_1D
7/27/2019 Danijela Blogs Sept27throughOctober11
http://slidepdf.com/reader/full/danijela-blogs-sept27throughoctober11 19/47
mCherry Expressing CellsGFP Photoactivation
mCherry
7/27/2019 Danijela Blogs Sept27throughOctober11
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Draq5 Nuclear Stain
Looking for mCherry Expressing Cells
CE39_Vagina_1D
mCherry
7/27/2019 Danijela Blogs Sept27throughOctober11
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Draq5 Nuclear Stain
Can we photoactivate GFP in mCherry expressing cells?
CE39_Vagina_1D
7/27/2019 Danijela Blogs Sept27throughOctober11
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297 pre PA
642 post PA
54% increase
279 pre PA
626 post PA
56% increase
prePA line
postPA line
mCherry
7/27/2019 Danijela Blogs Sept27throughOctober11
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Draq5 Nuclear Stain
Can we photoactivate GFP in mCherry expressing cells?
CE39_Vagina_1D
mCherry
l i
7/27/2019 Danijela Blogs Sept27throughOctober11
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Draq5 Nuclear Stain
Volume View: Click to Play
7/27/2019 Danijela Blogs Sept27throughOctober11
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474 pre PA
850 post PA
45% increase
Background
prePA line
postPA line
7/27/2019 Danijela Blogs Sept27throughOctober11
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Target Cells OptimizationCD3(neat)::Cy5 CE39_Vagina_1D
7/27/2019 Danijela Blogs Sept27throughOctober11
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Target Cells OptimizationCD3(neat)::Cy5 CE39_Vagina_1D
I purposely
overexposed
this image to
also show
the tick
epithelia of
this animal
and that
many CD3
cells are
found also in
here.
7/27/2019 Danijela Blogs Sept27throughOctober11
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Target Cells OptimizationCD3(neat)::Cy5
BFP
CE39_Vagina_3B
Same Area but Different Z
7/27/2019 Danijela Blogs Sept27throughOctober11
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Target Cells Optimization
CD11b::Zenon 647 Labeling CE39_Vagina_3B
7/27/2019 Danijela Blogs Sept27throughOctober11
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CD11b::Cy5
Connective Tissue in Blue
CE39_Vagina_3B
Target Cells Optimization
CE39 Vagina 3B
7/27/2019 Danijela Blogs Sept27throughOctober11
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BFP
CD3(neat)::Cy5
Draq5
BFP positive,
CD3 negative
cell with clearly
defined nuclei
as evident by
Draq5 staining
BFP negative,
CD3 positive
Cells
CE39_Vagina_3B
CE39 Vagina 3B
7/27/2019 Danijela Blogs Sept27throughOctober11
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BFP positive,
CD3 negative
cell with clearly
defined nuclei
as evident by
Draq5 staining
BFP negative,
CD3 positive
Cells
CE39_Vagina_3B
BFP
CD3(neat)::Cy5
Draq5
CE39 Vagina 3B
7/27/2019 Danijela Blogs Sept27throughOctober11
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BFP positive,
CD3 negative
cell with clearly
defined nuclei
as evident by
Draq5 staining
BFP negative,
CD3 positive
Cells
CE39_Vagina_3B
BFP
CD3(neat)::Cy5
Draq5
CE39 Vagina 3B
7/27/2019 Danijela Blogs Sept27throughOctober11
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BFP positive,
CD3 negative
cell with clearly
defined nuclei
as evident by
Draq5 staining
BFP negative,
CD3 positive
Cells
_ g _
BFP
CD3(neat)::Cy5
Draq5
CE39 Vagina 3B
7/27/2019 Danijela Blogs Sept27throughOctober11
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BFP positive,
CD3 negative
cell with clearly
defined nuclei
as evident by
Draq5 staining
_ g _
BFP
CD3(neat)::Cy5
Draq5
CE39_Vagina_3B
7/27/2019 Danijela Blogs Sept27throughOctober11
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BFP positive,
CD3 negative
cell with clearly
defined nuclei
as evident by
Draq5 staining
_ g _
BFP
CD3(neat)::Cy5
Draq5
CE39_Vagina_3B
7/27/2019 Danijela Blogs Sept27throughOctober11
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BFP positive,
CD3 negative
cell with clearly
defined nuclei
as evident by
Draq5 staining
BFP
CD3(neat)::Cy5
Draq5
CE39_Vagina_3B
7/27/2019 Danijela Blogs Sept27throughOctober11
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BFP positive,
CD3 negative
cell with clearly
defined nuclei
as evident by
Draq5 staining
BFP
CD3(neat)::Cy5
Draq5
CE39_Vagina_3B
7/27/2019 Danijela Blogs Sept27throughOctober11
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BFP positive,
CD3 negative
cell with clearly
defined nuclei
as evident by
Draq5 staining
Many BFP
negative, CD3
positive CellsBFP
CD3(neat)::Cy5
Draq5
CD3::Cy5
7/27/2019 Danijela Blogs Sept27throughOctober11
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Expected Cy5 Emission
Maxima is at 667nm
7/27/2019 Danijela Blogs Sept27throughOctober11
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ExpecteEmissio
610nm
Expecte
Maxima
Spectral Profile for
mCherry positive/CD3
negative cell whose
nucleus was stained with
Draq5
CD3/Alexa Fluor 647 Antibodies
7/27/2019 Danijela Blogs Sept27throughOctober11
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CD3 (1:5 dilution)
Alexa Fluor 647 (1:1000)
DRAQ5 Nuclear Stain
CD3 (1:10 dilution)
Alexa Fluor 647 (1:1000 dilution)
DRAQ5 Nuclear Stain
CD3 (1:50 dilution)
Alexa Fluor 647 (1:1000 dilution)
DRAQ5 Nuclear Stain
Secondar
Alexa Fluo
DRAQ5 N
CD3/Alexa Fluor 647 AntibodiesOptimization
All images were taken at same exposure/percent transmission and normalized.
CD3::AF647
7/27/2019 Danijela Blogs Sept27throughOctober11
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CD3::AF647Spectral Profile
Draq5
7/27/2019 Danijela Blogs Sept27throughOctober11
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Draq5Spectral Profile
::and DRAQ5
7/27/2019 Danijela Blogs Sept27throughOctober11
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and DRAQ5SpectralProfiles
• Note that when CD3 antibody is
used in 1:5 dilution and AF647 us
used at 1:1,000 dilution there is
absolutely no bleed-thru into thered channel.
• Also, note lower intensity of the
DRAQ5 in comparison to
CD3::AF647 peak which is logical
considering that we are using
very diluted nuclear stain.
• Both AF647 and DRAQ5 spectral
peaks fall in the desired rangeand it is possible to spectrally
differentiate between these two
Cy5 markers by this method.
After Spectral Unmixing
7/27/2019 Danijela Blogs Sept27throughOctober11
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After Spectral Unmixing
CD3 (1:5)::Alexa Fluor 647 (1:1,000)
DRAQ5 nuclear stain (1:20,000)
7/27/2019 Danijela Blogs Sept27throughOctober11
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After Spectral UnmixingCD3 (1:5)::Alexa Fluor 647 (1:1,000)
DRAQ5 nuclear stain (1:20,000)
merge