Use anti IgM with a fluorescent fused secondary antibody to test for membrane localization of the BCR complex. Use B-Cells as a control.
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Purpose
This will test whether or not the BCR complex will reach the cell membrane in HEK293 cells.
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Parts Needed
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
fused to a yellow alexa dye
B cells
from Velia
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Setup
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
EMPTY
EMPTY
EMPTY
EMPTY
EMPTY
EMPTY
EMPTY
EMPTY
EMPTY
EMPTY
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Expected Results
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.
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Protocol
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Microscopy Data
Microscopy Data:
Left: HEK 293 transfected with dummy DNA
Right: HEK 293 transfected with full BCR plasmids
Image ModifiedImage Modified
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Cytometry Data
Cytometry data were gated to remove non-cell data points.
