Group A: Membrane Localization

LilrB2

TRIAL 1

TRIAL 2

Results:

 LilrB2 Trial 1

Microscopy

Permeabilized	Non-permeabilized	Non-permeabilized stained control

Cytometry

Tube 2	Tube 3	Tube 4	Tube 5
Tube 8	Tube 9	Tube 10	Tube 11

Overlay of 4 and 5:

hef1a:mKate + dummy		hef1a:mKate + hef1a:LilrB2
	+		=

 

 

PirB

TRIAL 1

TRIAL 2

Results:

 PirB Trial 1

Microscopy

Control	Permeabilized	Non-permeabilized	LilrB2 Paper

Cytometry

 Gating

SSC-A vs FSC-A	FSC-H vs FSC-W	SSC-H vs SSC-W	Yellow vs Red

Tube 1	Tube 2	Tube 3	Tube 4
Tube 5	Tube 6	Tube 7	Tube 8

Analysis: 

Cytometry..

LilrB2 and PirB Trial 1: The plots for the mkate only and mkate + receptor cell populations are very similar. This is most probably due to the bleed through of the red channel into the yellow channel (the 'sensitivity' of the PMTs for the yellow channel was increased because the yellow fluorophore was not very bright).

Next steps:

• We are going to transfect the receptors without using a transfection marker (Trial 2).

Microscopy..

LilrB2 and PirB Trial 1: For both the receptors, there doesn't seem to be any clear localization of the receptors to the membrane. From the punctate appearance of the cells under the confocal, they seem to be significant localization of the receptors in some subcellular cytoplasmic structure. They may be making it to the membrane; however we are unable to discern that just from the images. Interestingly, this cytoplasmic localization seems to be apparent in the non-permeabilized cells which may mean that the fixing protocol is causing some kind of permeabilization in the cells. It may be of significance to note that in the LilrB2 paper (the one we are trying to replicate), they expressed the receptors under CMV (not hEF1a). Depending on the relative strengths of the promoters, the differing levels of expression (probably higher expression through hEF1a) might be causing the staining pattern that we are seeing.

Next steps:

• Perhaps staining live cells (to skip the fixation step) and hopefully conclude whether or not some of the receptor is making it to the membrane. (We are also going to do the beta amyloid binding experiment despite being unsure whether or not the receptor is making it to the membrane in hopes that it does bind and we can conclude that it is localizing.)
• Once we get the receptor-fluorescent protein fusions out of cloning, we can express them and look for localization. This will tell us whether or not the staining pattern that we're seeing is due to the protein expression or the staining.
• Clone the receptors into a different promoter: CMV or a TRE to determine if high expression levels are the problem. Having inducible expression of the receptors may be helpful later on in determining what the optimal level of receptor expression for the system.