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Untransfected

Control

Single Color

Control

Background Beta

Amyloid Binding

Background

Strep Binding

Beta Amyloid Binding

to Gantenerumab

Well 1

HEK293

Well 2

HEK293

hEF1a:eBFP 100ng

Dummy 400ng

Well 3

HEK293

hEF1a:eBFP 100ng

Dummy 400ng

aBeta 500 nM

Strep 10 ug/mL

Well 4

HEK293

hEF1a:eBFP 100ng

hEF1a:GMabH 100ng

hEF1a:GMabL 100ng

hEF1a:CD79A 100ng

hEF1a:CD79B 100ng

Strep 10 ug/mL

Well 5

HEK293

hEF1a:eBFP 100ng

hEF1a:GMabH 100ng

hEF1a:GMabL 100ng

hEF1a:CD79A 100ng

hEF1a:CD79B 100ng

aBeta 500 nM

Strep 10 ug/mL

Well 7

HEK293

Well 8

HEK293

hEF1a:eBFP 100ng

Dummy 400ng

Well 9

HEK293

hEF1a:eBFP 100ng

Dummy 400ng

aBeta 500 nM

Strep 10 ug/mL

Well 10

HEK293

hEF1a:eBFP 100ng

hEF1a:GMabH 100ng

hEF1a:GMabL 100ng

hEF1a:CD79A 100ng

hEF1a:CD79B 100ng

Strep 10 ug/mL

Well 11

HEK293

hEF1a:eBFP 100ng

hEF1a:GMabH 100ng

hEF1a:GMabL 100ng

hEF1a:CD79A 100ng

hEF1a:CD79B 100ng

aBeta 500 nM

Strep 10 ug/mL

Results:

Untransfected Control	Single Color Control	Background Beta Amyloid Binding	Background Strep Binding	Beta Amyloid Binding to Gantenerumab
Well 1 HEK293	Well 2 HEK293 hEF1a:eBFP 100ng Dummy 400ng	Well 3 HEK293 hEF1a:eBFP 100ng Dummy 400ng aBeta 500 nM Strep 10 ug/mL	Well 4 HEK293 hEF1a:eBFP 100ng hEF1a:GMabH 100ng hEF1a:GMabL 100ng hEF1a:CD79A 100ng hEF1a:CD79B 100ng Strep 10 ug/mL	Well 5 HEK293 hEF1a:eBFP 100ng hEF1a:GMabH 100ng hEF1a:GMabL 100ng hEF1a:CD79A 100ng hEF1a:CD79B 100ng aBeta 500 nM Strep 10 ug/mL
Image Modified	Image Modified	Image Modified	Image Modified	Image Modified
Image Modified	Image Modified	Image Modified	Image Modified	Image Modified

Expand
title Gating
1. BFP + 2. Image Modified

	BCR - AB +	BCR + AB +

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Expand
title Plate map
Untransfected Control Single Color Control Background Beta Amyloid Binding Background Strep Binding Beta Amyloid Binding to Gantenerumab   Well 1 HEK293 Well 2 HEK293 hEF1a:eBFP 100ng Dummy 400ng Well 3 HEK293 hEF1a:eBFP 100ng Dummy 400ng aBeta 500 nM Strep 10 ug/mL   Well 4 HEK293 hEF1a:eBFP 100ng hEF1a:GMabH 100ng hEF1a:GMabL 100ng hEF1a:CD79A 100ng hEF1a:CD79B 100ng Strep 10 ug/mL Well 5 HEK293 hEF1a:eBFP 100ng hEF1a:GMabH 100ng hEF1a:GMabL 100ng hEF1a:CD79A 100ng hEF1a:CD79B 100ng aBeta 500 nM Strep 10 ug/mL     Well 7 HEK293 Well 8 HEK293 hEF1a:eBFP 100ng Dummy 400ng Well 9 HEK293 hEF1a:eBFP 100ng Dummy 400ng aBeta 500 nM Strep 10 ug/mL   Well 10 HEK293 hEF1a:eBFP 100ng hEF1a:GMabH 100ng hEF1a:GMabL 100ng hEF1a:CD79A 100ng hEF1a:CD79B 100ng Strep 10 ug/mL Well 11 HEK293 hEF1a:eBFP 100ng hEF1a:GMabH 100ng hEF1a:GMabL 100ng hEF1a:CD79A 100ng hEF1a:CD79B 100ng aBeta 500 nM Strep 10 ug/mL

Results:

Untransfected

Control

Single Color

Control

Background Beta

Amyloid Binding

Background

Strep Binding

Beta Amyloid Binding

to Gantenerumab

Well 1

HEK293

Well 2

HEK293

hEF1a:eBFP 100ng

Dummy 400ng

Well 3

HEK293

hEF1a:eBFP 100ng

Dummy 400ng

aBeta 500 nM

Strep 10 ug/mL

 

Well 4

HEK293

hEF1a:eBFP 100ng

hEF1a:GMabH 100ng

hEF1a:GMabL 100ng

hEF1a:CD79A 100ng

hEF1a:CD79B 100ng

Strep 10 ug/mL

Well 5

HEK293

hEF1a:eBFP 100ng

hEF1a:GMabH 100ng

hEF1a:GMabL 100ng

hEF1a:CD79A 100ng

hEF1a:CD79B 100ng

aBeta 500 nM

Strep 10 ug/mL

 

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Expand
title Gating
1. BFP + 2. Well 3 HEK293 hEF1a:eBFP 100ng Dummy 400ng aBeta 500 nM Strep 10 ug/mL   Well 4 Strep 10 ug/mL Well 5 hEF1a:eBFP 100ng hEF1a:GMabH 100ng hEF1a:GMabL 100ng hEF1a:CD79A 100ng hEF1a:CD79B 100ng HEK293 Untransfected Control Single Color Control Background Beta Amyloid Binding Background Strep Binding Beta Amyloid Binding to Gantenerumab   Well 1 HEK293 Well 2 HEK293 hEF1a:eBFP 100ng Dummy 400ng HEK293 hEF1a:eBFP 100ng hEF1a:GMabH 100ng hEF1a:GMabL 100ng hEF1a:CD79A 100ng hEF1a:CD79B 100ng aBeta 500 nM Strep 10 ug/mL

BCR -

AB +

BCR +

AB +

 

 

 

Discussion:

 

 

 

Expand
title Progress
Cloning Transfection Staining Cytometry Analysis iGEM parts 07/13/15 07/15/15 07/15/15 07/16/15

Expand
title Background
Testing Gantenerumab binding to beta amyloid oligomers in the context of the B-Cell Receptor. Repeat of experiment done during iGEM.

Expand
title Approach
Testing beta amyloid binding to cells expressing the components of the B-Cell Receptor. To do this, we are going to treat cells with a saturating concentration of biotin-conjugated beta amyloid. Subsequently, we are going to treat with AlexaFluor 594 conjugated streptavidin. If beta-amyloid binds to the receptor, streptavidin can bind biotin and be detected via flow cytometry. We are controlling for eBFP bleed through into the red channel, background beta-amyloid binding and background streptavidin binding.

Expand
title Parts Needed
hEF1a:eBFP hEF1a:GMabH hEF1a:GmabL hEF1a:CD79A hEF1a:CD79B