Versions Compared

Old Version 30

changes.mady.by.user Unknown User (clrichar@mit.edu)

Saved on

compared with

New Version 31

changes.mady.by.user Unknown User (clrichar@mit.edu)

Saved on

Key

  • This line was added.
  • This line was removed.
  • Formatting was changed.

...

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

Well 1

B-Cells

Well 2

B-Cells

Anti IgM

Well 3

Permeablized B-Cells

Anti IgM

Well 4

HEK293

Well 5

HEK293

100 mKate

400 Dummy

Well 6

EMPTY

Well 7

HEK293

100 Gmab H

100 Gmab L

100 CD79A

100 CD79B

100 mKate

Anti IgM

Well 8 

HEK293

50 ng Gmab H

50 ng Gmab L

50 ng CD79A

50 ng CD79B

100 ng mKate

200 ng mKate

Anti IgM

Well 9

HEK293

25 ng Gmab H

25 ng Gmab L

25 ngCD79A

25 ng CD79B

100 ng mKate

300 ng Dummy DNA

Anti IgM

Well 10

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 11

EMPTY

 

Well 12

EMPTY

Well 13 

HEK293

100 Gmab H

100 Gmab L

100 CD79A

100 CD79B

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 16

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 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.

Protocol

 

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