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Use anti IgM with a fluorescent fused secondary antibody to test for membrane localization of the BCR complex.

This will test whether or not the BCR complex will reach the cell membrane in HEK293 cells.

pEXPR hEF1a: Gmab Light

pEXPR hEF1a: Gmab Heavy

pEXPR hEF1a: CD79A

pEXPR hEF1a: CD79B

ALSO NEED:

Anti IgM primary antibody

Fluorescently tagged yellow secondary antibody

B cells from Velia

PLATE 1

Well 1

B-Cells

Well 2

HEK293

Well 3

HEK293

Dummy DNA 1000 ng

Well 4

HEK293

mKate 200 ng

Dummy DNA 800 ng

Well 5 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 200 ng

CD79B 200 ng

mKate 200 ng


Well 6 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 100 ng

CD79B 100 ng

mKate 200 ng

Dummy DNA 200 ng

Well 7

B-Cells

Well 8 

HEK293

Well 9 

HEK293

Dummy DNA 1000 ng

 

Well 10 

HEK293

mKate 200 ng

Dummy DNA 800 ng


Well 11 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 200 ng

CD79B 200 ng

mKate 200 ng

 

Well 12 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 100 ng

CD79B 100 ng

mKate 200 ng

Dummy DNA 200 ng


Well 13 

B-Cells

Well 14 

HEK293

Well 15 

HEK293

Dummy DNA 1000 ng

 

Well 16 

HEK293

mKate 200 ng

Dummy DNA 800 ng

 

Well 17 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 200 ng

CD79B 200 ng

mKate 200 ng

 

Well 18 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 100 ng

CD79B 100 ng

mKate 200 ng

Dummy DNA 200 ng


Well 19 

B-Cells

Well 20

HEK293

Well 21 

HEK293

Dummy DNA 1000 ng

 

Well 22 

HEK293

mKate 200 ng

Dummy DNA 800 ng

 

Well 23 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 200 ng

CD79B 200 ng

mKate 200 ng

 

Well 24 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 100 ng

CD79B 100 ng

mKate 200 ng

Dummy DNA 200 ng


PLATE 2

Well 1

B-Cells

Well 2

HEK293

Well 3

HEK293

Dummy DNA 1000 ng

Well 4

HEK293

mKate 200 ng

Dummy DNA 800 ng

Well 5 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 200 ng

CD79B 200 ng

mKate 200 ng


Well 6 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 100 ng

CD79B 100 ng

mKate 200 ng

Dummy DNA 200 ng

Well 7

B-Cells

Well 8 

HEK293

Well 9 

HEK293

Dummy DNA 1000 ng

 

Well 10 

HEK293

mKate 200 ng

Dummy DNA 800 ng


Well 11 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 200 ng

CD79B 200 ng

mKate 200 ng

 

Well 12 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 100 ng

CD79B 100 ng

mKate 200 ng

Dummy DNA 200 ng


EMPTY

EMPTY

EMPTYEMPTYEMPTYEMPTY
EMPTYEMPTYEMPTYEMPTYEMPTYEMPTY

We expect to see clear yellow fluorescence around the blue stained nuclei indicating BCR localization to the HEK cell membrane. This will be corroborated by similar results with B cells.

 

Microscopy Data:

Left: HEK 293 transfected with dummy DNA

Right: HEK 293 transfected with full BCR plasmids

Cytometry data were gated to remove non-cell data points.

 

 

 

BCR localizes on membrane

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