Group B: Receptor Affinity to Beta-amyloid

Description:

In this group of experiments, we are aiming to test the affinity of our receptors, LilrB2 and PirB to beta-amyloid oligomers. To do that, we will have HEK293 cells express LilrB2 and PirB fused with YFP. We will then apply biotinylated beta-amyloid oligomers to the LilrB2 and PirB expressing cells. To test for binding, we are going to use the biotin-binding protein streptavidin covalently bonded to a fluorophore. We will look for co-localization of the fluorophore and the beta-amyloid. 

*We may potentially extend this experiment to trying to characterize the interaction between beta-amyloid and the receptors i.e. try to generate saturation plots and calculate dissociation constants.

Results

LilrB2

 LilrB2 Trial 1 Cytometry

Well 1 HEK293	Well 2 HEK293 eBFP Dummy	Well 3 HEK293 eBFP Dummy aBeta Strep	Well 4 HEK293 eBFP LilrB2 Strep	Well 5 HEK293 eBFP LilrB2 aBeta Strep

 LilrB Microscopy Trial 1

	Well 1 HEK293	Well 2 HEK293 aBeta Strep	Well 3 HEK293 eYFP Dummy	Well 4 HEK293 eYFP Dummy aBeta Strep	Well 5 HEK293 eYFP LilrB2 Strep	Well 6 HEK293 eYFP LilrB2 aBeta Strep
(a)

Untransfected stained	Transfected stained

 LilrB2 Trial 2 Cytometry

Transfection marker: tagBFP

Staining: biotinylated beta amyloid and strep-AF594

Well 1 HEK293 (unstained)	Well 2 HEK293 (stained)	Well 3 HEK293 eBFP Dummy aBeta Strep	Well 4 HEK293 eBFP LilrB2 Strep	Well 5 HEK293 eBFP LilrB2 aBeta Strep
	Lots of background..
Duplicates

PirB

 PirB Cytometry Trial 1

Well 1 HEK293	Well 2 HEK293 eBFP Dummy	Well 3 HEK293 eBFP Dummy aBeta Strep	Well 4 HEK293 eBFP PirB Strep	Well 5 HEK293 eBFP PirB aBeta Strep

eBFP + Dummy; aBeta + Strep	eBFP + PirB; aBeta + Strep	Overlay
MEAN: 171	MEAN: 1069

 PirB Microscopy Trial 1

Well 1 HEK293 Well 2 HEK293 eBFP Dummy Well 3 HEK293 eBFP Dummy aBeta Strep   Well 4 HEK293 eBFP PirB Strep Well 5 HEK293 eBFP PirB aBeta Strep   Well 6 Well 7 HEK293 aBeta Strep Well 8 HEK293 eBFP Dummy Well 9 HEK293 eBFP Dummy aBeta Strep   Well 10 HEK293 eBFP PirB Strep Well 11 HEK293 eBFP PirB aBeta Strep   Well 12

Before staining After staining sad cells after staining (evos picture - blue channel)   eBFP and PirB (aBeta + Strep) Blue Red Composite

 

 

 PirB Microscopy Trial 2

	Well 1 HEK293	Well 2 HEK293 aBeta Strep	Well 3 HEK293 eYFP Dummy	Well 4 HEK293 eYFP Dummy aBeta Strep	Well 5 HEK293 eYFP PirB Strep	Well 6 HEK293 eYFP PirB aBeta Strep
(a)
(b)

Untransfected stained	Transfected stained

Analysis

LilrB2

Trial 1: 

The cell population transfected with LilrB2 does not show any increase in red fluorescence which may indicate that LilrB2 does not bind abeta. However, from the picture taken of the cells before staining, it appears that the transfection efficiency for that well is very low making the reliability of the results form this trial very questionable. 

PirB

Cytometry Trial 1: 

The cell population transfected with PirB shows a marked increase in red fluorescence which indicates that PirB is in fact binding abeta. Moreover, when the distribution of red fluorescence of the transfected population (gated using the blue fluorescence distribution) is plotted, you see a shift in the distribution towards more red fluorescence and the mean increases from 171 in the PirB - population to 1069 in the PirB + population. 

It is interesting to note the "non-linear" distribution of red vs. blue in the PirB + population. We do not begin to see a significant increase in red fluorescence until we reach the more highly transfected cells. It would be interesting to plot mean blue vs. mean red to try to analyze this further. 

• Next steps: Microscopy! to try and confirm the results we are getting through cytometry