- Created by Unknown User (clrichar@mit.edu), last modified by Unknown User (asjm@mit.edu) on Oct 17, 2014 18:36
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 (actually, some papers say that it doesn't?) 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 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
| 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 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 |
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.
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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