- Created by Unknown User (clrichar@mit.edu), last modified by Unknown User (krbrink@mit.edu) on Sep 24, 2014 11:09
You are viewing an old version of this page. View the current version.
Compare with Current View Page History
« Previous Version 11 Next »
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
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 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
- No labels