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

Capstone Experiment to test Syk-TEV without Lyn.

hEF1a: Gmab H

hEF1a: Gmab L

hEF1a: CD79A

hEF1a: CD79B

TRE: Syk-TEV

hEF1a: rtTA

UAS: mKate

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

We expect the tango cleavage system to have virtually no difference between active and inactive receptors because the BCR requires(question) Lyn to recruit Syk.

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

 

 

 

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

Capstone Experiment to test Syk-TEV without Lyn.

hEF1a: Gmab H

hEF1a: Gmab L

hEF1a: CD79A

hEF1a: CD79B

TRE: Syk-TEV

hEF1a: rtTA

UAS: mKate

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 1Well 2Well 3Well 4Well 5Well 6

untransfected

(calibration)

eBFP bleed through controlleaky mKate control

TEVp control

(nonspecific G4 cleavage)

TEVp control

(nonspecific G4 cleavage)

TEVp control

(nonspecific G4 cleavage)

HEK293

 

 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 7Well 8Well 9Well 10Well 11Well 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 13Well 14Well 15Well 16Well 17Well 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 19Well 20Well 21Well 22Well 23Well 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

We expect the tango cleavage system to have virtually no difference between active and inactive receptors because the BCR requires(question) Lyn to recruit Syk.

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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