danijela blogs sept27throughoctober11

7/27/2019 Danijela Blogs Sept27throughOctober11

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Danijela BlogsSeptember 24-October1



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



BFP Expressing CellsGFP PhotoactivationSpectral Imaging



BFP

Draq5 Nuclear Stain

Looking for BFP Expressing Cells

CE39_Vagina_1D



BFP

Draq5 Nuclear Stain


CE39_Vagina_1D



BFP

Draq5 Nuclear Stain

Volume View: Click to Play



BFP

Draq5 Nuclear Stain


CE39_Vagina_1D

BFP



BFP

Draq5 Nuclear Stain


BFP



BFP

Draq5 Nuclear Stain


CE39_Vagina_1D

BFP



BFP

Draq5 Nuclear Stain


BFP



BFP

Draq5 Nuclear Stain

Can we photoactivate GFP in BFP expressing cells?

CE39_Vagina_1D



166 pre PA

188 post PA

12% increase

Background

prePA line

postPA line

BFP



BFP

Draq5 Nuclear Stain


CE39_Vagina_1D



136 pre PA

171 post PA

21% increase

Background

prePA line

postPA line

124 pre PA

238 post PA

48% increaseCE39_Vagina_1D

BFP



BFP

Draq5 Nuclear Stain


CE39_Vagina_1D



113 pre PA

209 post PA

46% increase

Background

prePA line

postPA line

Spectral imaging



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



mCherry Expressing CellsGFP Photoactivation

mCherry



Draq5 Nuclear Stain

Looking for mCherry Expressing Cells

CE39_Vagina_1D

mCherry



Draq5 Nuclear Stain

Can we photoactivate GFP in mCherry expressing cells?

CE39_Vagina_1D



297 pre PA

642 post PA

54% increase

279 pre PA

626 post PA

56% increase

prePA line

postPA line

mCherry



Draq5 Nuclear Stain

Can we photoactivate GFP in mCherry expressing cells?

CE39_Vagina_1D

mCherry

l i



Draq5 Nuclear Stain




474 pre PA

850 post PA

45% increase

Background

prePA line

postPA line



Target Cells OptimizationCD3(neat)::Cy5 CE39_Vagina_1D



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.



Target Cells OptimizationCD3(neat)::Cy5

BFP

CE39_Vagina_3B

Same Area but Different Z



Target Cells Optimization

CD11b::Zenon 647 Labeling CE39_Vagina_3B



CD11b::Cy5

Connective Tissue in Blue

CE39_Vagina_3B

Target Cells Optimization

CE39 Vagina 3B



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



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



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



BFP positive,

CD3 negative

cell with clearly

defined nuclei

as evident by

Draq5 staining

_ g _

BFP

CD3(neat)::Cy5

Draq5

CE39_Vagina_3B



BFP positive,

CD3 negative

cell with clearly

defined nuclei

as evident by

Draq5 staining

BFP

CD3(neat)::Cy5

Draq5

CE39_Vagina_3B



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



Expected Cy5 Emission

Maxima is at 667nm



ExpecteEmissio

610nm

Expecte

Maxima

Spectral Profile for

mCherry positive/CD3

negative cell whose

nucleus was stained with

Draq5

CD3/Alexa Fluor 647 Antibodies



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



CD3::AF647Spectral Profile

Draq5



Draq5Spectral Profile

::and DRAQ5



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



After Spectral Unmixing

CD3 (1:5)::Alexa Fluor 647 (1:1,000)

DRAQ5 nuclear stain (1:20,000)



After Spectral UnmixingCD3 (1:5)::Alexa Fluor 647 (1:1,000)

DRAQ5 nuclear stain (1:20,000)

merge

danijela blogs sept27throughoctober11

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