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title	Test 2

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title	Description

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

title	Purpose

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

Expand

title	Parts 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

title	Setup

PLATE 1

Well 1

B-Cells

Well 2

HEK293

Well 3

HEK293

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

B-Cells

Well 7

B-Cells

Well 8

HEK293

Well 9

HEK293

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

B-Cells

Well 13

B-Cells

Well 14

HEK293

Well 15

HEK293

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

B-Cells

Well 19

B-Cells

Well 20

HEK293

Well 21

HEK293

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

B-Cells

PLATE 2

Well 1 B-Cells	Well 2 HEK293	Well 3 HEK293	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 B-Cells
Well 7 B-Cells	Well 8 HEK293	Well 9 HEK293	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 B-Cells
EMPTY	EMPTY	EMPTY	EMPTY	EMPTY	EMPTY
EMPTY	EMPTY	EMPTY	EMPTY	EMPTY	EMPTY

Expand

title	Expected 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

title	Protocol

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

title	Data

Expand

title	Analysis

EMPTY

Expand

title	Test 23

Expand

title	Description

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

title	Purpose

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

Expand

title	Parts 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

Well 5

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 200 ng

CD79B 200 ng

mKate 200 ng

Well 10

HEK293

mKate 200 ng

Dummy DNA 800 ng

Expand

title	Setup

PLATE 1

L 200 ng H 200 ng 200 ng 200 ng 200 ngWell 14

Well 1

B-Cells

Well 2

B-Cells

Anti IgMHEK293

Well 3

HEK293

Dummy DNA

Anti IgM

Well 4

EMPTY

Well 5

HEK293

mKate 200 ng

Dummy DNA 800 ng

Well 5

EMPTY

Well 6

EMPTY

Well 7

HEK293

Gmab

H

Gmab

L

CD79A

CD79B

mKate

Well 6

B-Cells

Well 7

B-Cells

Well 8

HEK293

Well 9

HEK293

Well 10

HEK293

mKate 200 ng Gmab H

Dummy DNA 800 ng

Well 11

HEK293

200 ng Gmab L 200 ngGmab

H 200 ng CD79A

CD79A 200 ng CD79B

CD79B 200 ng mKate 200 ng

Anti IgM

Well 12

B-Cells

Well 13

B-Cells

9

HEK293

Well 15

HEK293

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

B-Cells

Well 19

B-Cells

Well 20

HEK293

Well 21

HEK293

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

B-Cells

PLATE 2

Well 1

B-Cells

Well 2

HEK293

Well 3

HEK293

Well 4

HEK293

mKate 200 ng

Dummy DNA 800 ng

Well 6

B-Cells

Well 7

B-Cells

Well 8

HEK293

Well 9

HEK293

Well 11

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 200 ng

CD79B 200 ng

mKate 200 ng

Well 12

B-Cells

100 ng Gmab H

100 ng Gmab L

100 ngCD79A

100 ng CD79B

400 ng Dummy DNA

200 ng mKate

Anti IgM

Well 10

HEK293

50 ng Gmab H

50 ng Gmab L

50 ng CD79A

50 ng CD79B

600 ng Dummy DNA

200 ng mKate

Anti IgM

Well 11

EMPTY

Well 12

EMPTY

Well 13

EMPTY

Well 14

EMPTY

Well 15

EMPTY

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

EMPTY

EMPTY

EMPTY

Expand

title	Expected 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

title	Protocol

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

title	Data

Expand

title	Analysis

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