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title	Test 1

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title	Description

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

Capstone Experiment to test Syk-TEV without Lyn.

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title	Parts Needed

hEF1a: Gmab H

hEF1a: Gmab L

hEF1a: CD79A

hEF1a: CD79B

TRE: Syk-TEV

hEF1a: rtTA

UAS: mKate

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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 20

HEK293

Well 21

HEK293

Well 22

HEK293

Well 23

HEK293

Well 24

HEK293

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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 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

Expand

title	Test 1

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

Expand

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 20

HEK293

Well 21

HEK293

Well 22

HEK293

Well 23

HEK293

Well 24

HEK293

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

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A

CD79B

TRE: Syk-TEVp

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A

CD79B

TRE: Syk-TEVp

UAS: mKate

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A-TCS-G4

CD79B

UAS: mKate

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A

CD79B-TCS-G4

UAS: mKate

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A-TCS-G4

CD79B-TCS-G4

UAS: mKate

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

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A-TCS-G4

CD79B

TRE: Syk-TEVp

UAS:mKate

10 nM dox

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A-TCS-G4

CD79B

TRE: Syk-TEVp

UAS:mKate

10 nM dox

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A-TCS-G4

CD79B

TRE: Syk-TEVp

UAS:mKate

10 nM dox

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A-TCS-G4

CD79B

TRE: Syk-TEVp

UAS:mKate

10 nM dox

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A-TCS-G4

CD79B

TRE: Syk-TEVp

UAS:mKate

10 nM dox

Well 13

Well 14

Well 15

Well 16

Well 17

Well 18

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A

CD79B-TCS-G4

TRE: Syk-TEVp

UAS:mKate

10 nM dox

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A

CD79B-TCS-G4

TRE: Syk-TEVp

UAS:mKate

10 nM dox

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A

CD79B-TCS-G4

TRE: Syk-TEVp

UAS:mKate

10nM dox

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A

CD79B-TCS-G4

TRE: Syk-TEVp

UAS:mKate

10 nM dox

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A

CD79B-TCS-G4

TRE: Syk-TEVp

UAS:mKate

10 nM dox

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A

CD79B-TCS-G4

TRE: Syk-TEVp

UAS:mKate

10 nM dox

Well 19

Well 20

Well 21

Well 22

Well 23

Well 24

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A-TCS-G4

CD79B-TCS-G4

TRE: Syk-TEVp

UAS:mKate

10 nM dox

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A-TCS-G4

CD79B-TCS-G4

TRE: Syk-TEVp

UAS:mKate

10 nM dox

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A-TCS-G4

CD79B-TCS-G4

TRE: Syk-TEVp

UAS:mKate

10nM dox

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A-TCS-G4

CD79B-TCS-G4

TRE: Syk-TEVp

UAS:mKate

10 nM dox

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A-TCS-G4

CD79B-TCS-G4

TRE: Syk-TEVp

UAS:mKate

10 nM dox

HEK293

hEF1a: eBFP

hEF1a: rtTA

GmabH

GmabL

CD79A-TCS-G4

CD79B-TCS-G4

TRE: Syk-TEVp

UAS:mKate

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 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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