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Expand
titleTest 3
Expand
titleDescription

Use anti IgM with a fluorescent fused secondary antibody to test for membrane localization of the BCR complex. Use B-Cells as a control.

Expand
titlePurpose

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

Expand
titleParts Needed

pEXPR hEF1a: Gmab Light

pEXPR hEF1a: Gmab Heavy

pEXPR hEF1a: CD79A

pEXPR hEF1a: CD79B

ALSO NEED:

Anti IgM primary antibody fused to a yellow alexa dye

B cells

Expand
titleSetup

PLATE 1

Well 1

EMPTY

Well 2

EMPTY

Well 3

HEK293

Well 4

HEK293

Dummy DNA 1000 ng

Well 5

HEK293

Gmab L 250 ng

Gmab H 250 ng

CD79A 250 ng

CD79B 250 ng


Well 6

EMPTY

Well 7

EMPTY

Well 8 

EMPTY

Well 9

EMPTY

Well 10

EMPTY


Well 11

EMPTY

 

Well 12

EMPTY

EMPTY

EMPTY

EMPTYEMPTYEMPTYEMPTY
EMPTYEMPTYEMPTYEMPTYEMPTYEMPTY
Expand
titleExpected Results

We expect to see clear yellow fluorescence around the blue stained nuclei indicating BCR localization to the HEK cell membrane. Moreover Transfection efficiency as measured by our mKate transfection marker should correlate to anti IgM fluorescence in the flow cytometer. B-Cells should act as a positive control for this experiment.

Expand
titleProtocol

calibrate cytometer with wells2/8

then run remaining tubes sequentially

image well 1 b-cells alt 7

image well 3 stained HEK293 cells with no DNA alt 9

image well 4 stained HEK293 cells withmkate + Dummy DNA alt 10

image well 5 stained HEK293 with BCR alt 11

image well 13 permeablized b-cells alt 19

image well 15 permeablized HEK293 with no DNA alt 21

image well 16 permeablized HEK293 with mkate+dummy alt 22

image well 17 permeablized HEK293 with BCR alt 23

Expand
titleData
Expand
titleB cell gating (8/1/2014)

Unstained B cells (8/1/2014)Stained B cells (8/1/2014)
Expand
titleHEK293 BCR gating (8/7/2014)

HEK293 dummy DNA stained (8/7/2014)HEK293 stained BCR (8/7/2014)
Image AddedImage Added
Expand
titleAnalysis

 

Expand
titleTest 4
Expand
titleDescription

Use anti IgM with a fluorescent fused secondary antibody to test for membrane localization of the BCR complex. Use B-Cells as a control. 

Expand
titlePurpose

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

Expand
titleParts Needed

pEXPR hEF1a: Gmab Light

pEXPR hEF1a: Gmab Heavy

pEXPR hEF1a: CD79A

pEXPR hEF1a: CD79B

ALSO NEED:

Anti IgM primary antibody fused to a yellow alexa dye

B cells

Expand
titleSetup

PLATE 1

9100 100 100 100 400 Dummy DNA 1050 50 50 CD79A50 CD79B600 200 ng mKate

Well 1

B-Cells

Well 2

B-Cells

Anti IgM

Well 3

HEK293

Dummy DNA

Permeablized B-Cells

Anti IgM

Well 4

EMPTYHEK293

Well 5

HEK293

100 mKate

400 DummyEMPTY

Well 6

EMPTY

Well 7

B-Cells

Well 8

B-Cells

Anti IgM

Well 9

Permeablized B-Cells

Anti IgM

Well 10

HEK293

Well 11

HEK293

100 mKate

400 Dummy

Well 12

EMPTY

Well 13

HEK293

Gmab H

Gmab L

CD79A

CD79B

mKate

Well 8 

HEK293

200 ng 100 Gmab H200 ng

100 Gmab L

200 ng 100 CD79A200 ng

100 CD79B

200 ng 100 mKate

Anti IgM

Well

14 

HEK293

50 ng Gmab H

50 ng Gmab L

50 ng CD79A

50 ng CD79B

100 ng

mKate

200 ng mKate

Anti IgM

Well

15

HEK293

25 ng Gmab H

25 ng Gmab L

25 ngCD79A

25 ng

CD79B

100 ng

mKate

300 ng Dummy DNA

Anti IgM

Well 1116

EMPTY

 

Well 12

EMPTY

Well 13 

EMPTY

Well 14 

EMPTY

Well 15

EMPTY

HEK293

12.5 ng Gmab H

12.5 ng Gmab L

12.5 ng CD79A

12.5 ng CD79B

100 ng mKate

350 ng Dummy DNA

Anti IgM

Well 16

EMPTY

 

Well 17

EMPTY

 

Well 18

EMPTY

Well 19 

EMPTY

Well 20

EMPTY

Well 21

EMPTY

Well 22

EMPTY

 

Well 23

EMPTY

 

Well 24 

EMPTY

Expand
titleExpected Results

We expect to see clear yellow fluorescence around the blue stained nuclei indicating BCR localization to the HEK cell membrane. Moreover Transfection efficiency as measured by our mKate transfection marker should correlate to anti IgM fluorescence in the flow cytometer. B-Cells should act as a positive control for this experiment.

HEK293

100 Gmab H

100 Gmab L

100 CD79A

100 CD79B

100 mKate

Anti IgM

Well 20

HEK293

50 ng Gmab H

50 ng Gmab L

50 ng CD79A

50 ng CD79B

100 ng mKate

200 ng mKate

Anti IgM

Well 21

HEK293

25 ng Gmab H

25 ng Gmab L

25 ngCD79A

25 ng CD79B

100 ng mKate

300 ng Dummy DNA

Anti IgM

Well 22

HEK293

12.5 ng Gmab H

12.5 ng Gmab L

12.5 ng CD79A

12.5 ng CD79B

100 ng mKate

350 ng Dummy DNA

Anti IgM

 

Well 23

EMPTY

 

Well 24 

EMPTY

 

 

Expand
titleProtocol

 

Expand
titleProtocol

calibrate cytometer with wells2/8

then run remaining tubes sequentially

image well 1 b-cells alt 7

image well 3 stained HEK293 cells with no DNA alt 9

image well 4 stained HEK293 cells withmkate + Dummy DNA alt 10

image well 5 stained HEK293 with BCR alt 11

image well 13 permeablized b-cells alt 19

image well 15 permeablized HEK293 with no DNA alt 21

image well 16 permeablized HEK293 with mkate+dummy alt 22

image well 17 permeablized HEK293 with BCR alt 23

Expand
titleData

 

Expand
titleAnalysis