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title	Test 1

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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.Tests the effectiveness of a Syk-TEV cofactor in the tango system with no transfected Lyn, only possible endogenous Lyn.

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title	Purpose

This will test whether or not the BCR complex will reach the cell membrane in HEK293 cellsCapstone Experiment to test Syk-TEV without Lyn.

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title	Parts Needed

pEXPR hEF1a: Gmab LightHpEXPR

hEF1a: Gmab HeavyL

pEXPR hEF1a: CD79A

pEXPR hEF1a: CD79BALSO

NEEDTRE:

Anti IgM primary antibody fused to a yellow alexa dye

Syk-TEV

hEF1a: rtTA

UAS: mKateB cells

PLATE 1

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title	Setup

4

mKate 200 ng

Dummy DNA 800 ng 5

Gmab L 200 ng

Gmab H 200 ng

CD79A 200 ng

CD79B 200 ng

mKate 200 ng

Well 1

EMPTYHEK293

Well 2

HEK293

Well 3

HEK293

Well 4

HEK293

Well 5

HEK293

Well 6

HEK293Dummy DNA 1000 ng

Well

7

HEK293

Well 8

HEK293

Well

9

HEK293

Well 10

HEK293

Well 11

HEK293

Well 12

HEK293

Well 13

HEK293

Well 14

HEK293

Well 15

HEK293

Well 16

HEK293

Well 17

HEK293

Well 18

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

EMPTY

Well 8

HEK293

Well 919

HEK293

Dummy DNA 1000 ng

Well 20

HEK293

Well 21

HEK293

Well 1022

HEK293

Well 23

HEK293

mKate 200 ng

Dummy DNA 800 ng

Well 1124

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

EMPTY

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

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title	Expected Results

We expect the tango cleavage system to have virtually no difference between active and inactive receptors because the BCR requires (actually, some papers say that it doesn't?) Lyn to recruit Syk.

Expand

title	Protocol

Day1

Seed the cells on a plastic plate using splitting cells and seeding plates protocol

Day2

Transfect the cells using either the lipo2k protocol

Day3

add a dox ladder

Day4

do standard cytometry prep

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title	Microscopy Data

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title	Cytometry Data

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title	Analysis

B-Cells of any kind were unavailable for use as a positive control for this experiment. Moreover the mKate transfection marker in our control did not show as much red fluorescence as was expected. The cytometry data remains inconclusive but the BCR plots suggest that there was BCR localization. Moreover the microscopy clearly shows membrane localization of the BCR in both permeablized and non-permeablized cells. This experiment will be repeated once B-Cells Become available. Additionally in this experiment no untransfected unstained control was planned into the plate and thus we had to collect one on very short notice. For the next experiment this control will be planned into the experiment. A final problem with this experiment was a mix up in sample labels that resulted in a mislabeling of cytometry samples. The data however was sufficiently different for the samples to be re sorted into their original labels. Due to the various problems and limitations in this initial experiment there will be a repeat of this experiment done as soon as B-Cells become available as a positive control.

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title	Test 2

Expand

title	Description

Tests the effectiveness of a Syk-TEV cofactor in the tango system with no transfected Lyn, only possible endogenous Lyn.

Expand

title	Purpose

Capstone Experiment to test Syk-TEV without Lyn.

Expand

title	Parts Needed

hEF1a: Gmab H

hEF1a: Gmab L

hEF1a: CD79A

hEF1a: CD79B

TRE: Syk-TEV

hEF1a: rtTA

UAS: mKate

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

Well 12

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 100 ng

CD79B 100 ng

mKate 200 ng

Dummy DNA 200 ng

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title	Setup

Well 1

HEK293

Well 2

HEK293

Well 3

HEK293

Well 4

HEK293

Well 5

HEK293

Well 6

HEK293

Well 7

HEK293

Well 8

HEK293

Well 9

HEK293

Well 10

HEK293

Well 11

HEK293

Well 12

HEK293

Well 13

HEK293

Well 14

HEK293

Well 15

HEK293

Well 16

HEK293

Well 17

HEK293

Well 18

HEK293

Well 19

HEK293

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

EMPTY

Well 20

HEK293

Well 21

HEK293

Dummy DNA 1000 ng

Well 22

HEK293

Well 23

HEK293

Well 2224

HEK293

mKate 200 ng

Dummy DNA 800 ngWell 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

Well 1

Well 2

Well 3

Well 4

Well 5

Well 6

untransfected

(calibration)

eBFP bleed through control

leaky mKate control

TEVp control

(nonspecific G4 cleavage)

TEVp control

(nonspecific G4 cleavage)

TEVp control

(nonspecific G4 cleavage)

HEK293

PLATE 2

Well 1

EMPTY

Well 2

HEK293

Well 3

HEK293

Dummy DNA 1000 ng

Well 4

HEK293

mKate 200 ng

Dummy DNA 800 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

EMPTY

Well 8

HEK293

Well 9

HEK293

Dummy DNA 1000 ng

Well 11

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 200 ng

CD79B 200 ng

mKate 200 ng

EMPTY

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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.

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title	Protocol

Day 1

Cells were seeded using the Gelatin Treatment for Glass Plates protocol (1 mL/well was used) for plate 1. For plate 2 the standard Splitting Cells & seeding plates protocol was used.

Day 2

All cells were transfected based on the above plate map using the Transfection (Lipofectamine 2000) protocol.

Day 3

Cells were left to grow

Day 4

Plate two was run through the flow cytometer. They were prepared using the Immunostaining for Flow Cytometry protocol.

Plate one was used for fluorescent microscopy. It was prepared with the Immunostaining for Fluorescent microscopy protocol. Wells 1-12 were permeablized with Triton-X100 while wells 13-24 were left with their membranes intact.

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title	Microscopy Data

Microscopy Data:

Left: HEK 293 transfected with dummy DNA

Right: HEK 293 transfected with full BCR plasmids

Image RemovedImage Removed

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title	Cytometry Data

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

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title	Negative/negative control

Image Removed

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title	Untransfected HEK 293

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title	Transfected w/ Dummy DNA

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title	Transfected w/ mKate only

Image RemovedImage Removed

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title	Transfected w/ BCR Plasmids

Image RemovedImage Removed

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title	Analysis

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A

100 ug CD79B

100 ug TRE: Syk-TEVp

100 ug Dummy

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A

100 ug CD79B

100 ug TRE: Syk-TEVp

100 ug UAS: mKate

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A-TCS-G4

100 ug CD79B

100 ug UAS: mKate

100 ug Dummy

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A

100 ug CD79B-TCS-G4

100 ug UAS: mKate

100 ug Dummy

100 ug HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A-TCS-G4

100 ug CD79B-TCS-G4

100 ug UAS: mKate

100 ug Dummy

Well 7

Well 8

Well 9

Well 10

Well 11

Well 12

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A-TCS-G4

100 ug CD79B

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

10 nM dox

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

50 ug GmabH

50 ug GmabL

50 ug CD79A-TCS-G4

50 ug CD79B

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

200 ug Dummy

10 nM dox

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

25 ug GmabH

25 ug GmabL

25 ug CD79A-TCS-G4

25 ug CD79B

100 ug : Syk-TEVp

100 ug UAS:mKate

300 ug Dummy

10 nM dox

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

12.5 ug GmabH

12.5 ug GmabL

12.5 ug CD79A-TCS-G4

12.5 ug CD79B

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

350 ug Dummy

10 nM dox

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

6.25 ug GmabH

6.25 ug GmabL

6.25 ug CD79A-TCS-G4

6.25 ug CD79B

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

375 ug Dummy

10 nM dox

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

3.125 ug GmabH

3.125 ug GmabL

3.125 ug CD79A-TCS-G4

3.125 ug CD79B

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

387.5 ug Dummy

10 nM dox

Well 13

Well 14

Well 15

Well 16

Well 17

Well 18

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A

100 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

10 nM dox

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

50 ug GmabH

50 ug GmabL

50 ug CD79A

50 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

200 ug Dummy

10 nM dox

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

25 ug GmabH

25 ug GmabL

25 ug CD79A

25 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

300 ug Dummy

10nM dox

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

12.5 ug GmabH

12.5 ug GmabL

12.5 ug CD79A

12.5 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

350 ug Dummy

10 nM dox

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

6.25 ug GmabH

6.25 ug GmabL

6.25 ug CD79A

6.25 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

375 ug Dummy

10 nM dox

HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

3.125 ug GmabH

3.125 ug GmabL

3.125 ug CD79A

3.125 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

387.5 ug Dummy

10 nM dox

Well 19

Well 20

Well 21

Well 22

Well 23

Well 24

100 ug HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

100 ug GmabH

100 ug GmabL

100 ug CD79A-TCS-G4

100 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

10 nM dox

100 ugHEK293

100 ughEF1a: eBFP

100 ughEF1a: rtTA

50 ug GmabH

50 ug GmabL

50 ug CD79A-TCS-G4

50 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

200 ug Dummy

10 nM dox

100 ug HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

25 ug GmabH

25 ug GmabL

25 ug CD79A-TCS-G4

25 ug CD79B-TCS-G4

100 ugTRE: Syk-TEVp

100 ug UAS:mKate

300 ug Dummy

10nM dox

100 ug HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

12.5 ug GmabH

12.5 ug GmabL

12.5 ug CD79A-TCS-G4

12.5 ug CD79B-TCS-G4

100 ugTRE: Syk-TEVp

100 ugUAS:mKate

350 ug Dummy

10 nM dox

100 ug HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

6.25 ug GmabH

6.25 ug GmabL

6.25 ug CD79A-TCS-G4

6.25 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

375 ug Dummy

10 nM dox

100 ug HEK293

100 ug hEF1a: eBFP

100 ug hEF1a: rtTA

3.125 ug GmabH

3.125 ug GmabL

3.125 ug CD79A-TCS-G4

3.125 ug CD79B-TCS-G4

100 ug TRE: Syk-TEVp

100 ug UAS:mKate

387.5 ug Dummy

10 nM dox

Expand

title	Expected Results

We expect the tango cleavage system to have virtually no difference between active and inactive receptors because the BCR requires (actually, some papers say that it doesn't?) Lyn to recruit Syk.

Expand

title	Protocol

Day1

Seed the cells on a plastic plate using splitting cells and seeding plates protocol

Day2

Transfect the cells using either the lipo2k protocol

Day3

add a dox ladder

Day4

do standard cytometry prep

Expand

title	Microscopy Data

Expand

title	Cytometry Data

Expand

title	Analysis

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