Well 1
Well 2
Well 3
Well 4
Well 5
Well 6

untransfected

(calibration)

eBFP bleed throngh controlleaky mKate control

TEVp control

(nonspecific G4 cleavage)

TEVp control

(nonspecific G4 cleavage)

TEVp control

(nonspecific G4 cleavage)

HEK293

 

 HEK293

100 ng hEF1a: mKate

100 ng hEF1a: rtTA

100 ng GmabH

100 ng GmabL

100 ng CD79A

100 ng CD79B

100 ng TRE: Syk-TEVp

100 ng Lyn

 

HEK293

100 ng hEF1a: mKate

100 ng hEF1a: rtTA

100 ng GmabH

100 ng GmabL

100 ng CD79A

100 ng CD79B

100 ng TRE: Syk-TEVp

100 ng UAS: eYFP

100 ng Lyn

HEK293

100 ng hEF1a: mKate

100 ng hEF1a: rtTA

100 ng GmabH

100 ng GmabL

100 ng CD79A-TCS-G4

100 ng CD79B

100 ng UAS: eYFP

 

HEK293

100 ng hEF1a: mKate

100 ng hEF1a: rtTA

100 ng GmabH

100 ng GmabL

100 ng CD79A

100 ng CD79B-TCS-G4

100 ng UAS: eYFP

 

100 ng HEK293

100 ng hEF1a: mKate

100 ng hEF1a: rtTA

100 ng GmabH

100 ng GmabL

100 ng CD79A-TCS-G4

100 ng CD79B-TCS-G4

100 ng UAS: eYFP

 

Well 7Well 8Well 9Well 10Well 11Well 12

HEK293

100 ng hEF1a: mKate

100 ng hEF1a: rtTA

100 ng GmabH

100 ng GmabL

100 ng CD79A-TCS-G4

100 ng CD79B

100 ng TRE: Syk-TEVp

100 ng UAS: eYFP
100 ng Lyn

10 nM dox

 

HEK293

100 ng hEF1a: mKate

100 ng hEF1a: rtTA

50 ng GmabH

50 ng GmabL

50 ng CD79A-TCS-G4

50 ng CD79B

100 ng TRE: Syk-TEVp

100 ng UAS: eYFP
100 ng Lyn

10 nM dox

HEK293

100 ng hEF1a: mKate

100 ng hEF1a: rtTA

25 ng GmabH

25 ng GmabL

25 ng CD79A-TCS-G4

25 ng CD79B

100 ng : Syk-TEVp

100 ng UAS: eYFP
100 ng Lyn

10 nM dox

HEK293

100 ng hEF1a: mKate

100 ng hEF1a: rtTA

12.5 ng GmabH

12.5 ng GmabL

12.5 ng CD79A-TCS-G4

12.5 ng CD79B

100 ng TRE: Syk-TEVp

100 ng UAS: eYFP
100 ng Lyn

10 nM dox

HEK293

100 ng hEF1a: mKate

100 ng hEF1a: rtTA

6.25 ng GmabH

6.25 ng GmabL

6.25 ng CD79A-TCS-G4

6.25 ng CD79B

100 ng TRE: Syk-TEVp

100 ng UAS: eYFP
100 ng Lyn

10 nM dox

HEK293

100 ng hEF1a: mKate

100 ng hEF1a: rtTA

3.125 ng GmabH

3.125 ng GmabL

3.125 ng CD79A-TCS-G4

3.125 ng CD79B

100 ng TRE: Syk-TEVp

100 ng UAS: eYFP
100 ng Lyn

10 nM dox

Well 13Well 14Well 15Well 16Well 17Well 18

HEK293

100 ng hEF1a: mKate

100 ng hEF1a: rtTA

100 ng GmabH

100 ng GmabL

100 ng CD79A

100 ng CD79B-TCS-G4

100 ng TRE: Syk-TEVp

100 ng UAS: eYFP
100 ng Lyn

10 nM dox

HEK293

100 ng hEF1a: mKate

100 ng hEF1a: rtTA

50 ng GmabH

50 ng GmabL

50 ng CD79A

50 ng CD79B-TCS-G4

100 ng TRE: Syk-TEVp

100 ng UAS: eYFP
100 ng Lyn

10 nM dox

HEK293

100 ng hEF1a: mKate

100 ng hEF1a: rtTA

25 ng GmabH

25 ng GmabL

25 ng CD79A

25 ng CD79B-TCS-G4

100 ng TRE: Syk-TEVp

100 ng UAS: eYFP
100 ng Lyn

10nM dox

HEK293

100 ng hEF1a: mKate

100 ng hEF1a: rtTA

12.5 ng GmabH

12.5 ng GmabL

12.5 ng CD79A

12.5 ng CD79B-TCS-G4

100 ng TRE: Syk-TEVp

100 ng UAS: eYFP
100 ng Lyn

10 nM dox

HEK293

100 ng hEF1a: mKate

100 ng hEF1a: rtTA

6.25 ng GmabH

6.25 ng GmabL

6.25 ng CD79A

6.25 ng CD79B-TCS-G4

100 ng TRE: Syk-TEVp

100 ng UAS: eYFP
100 ng Lyn

10 nM dox

HEK293

100 ng hEF1a: mKate

100 ng hEF1a: rtTA

3.125 ng GmabH

3.125 ng GmabL

3.125 ng CD79A

3.125 ng CD79B-TCS-G4

100 ng TRE: Syk-TEVp

100 ng UAS: eYFP
100 ng Lyn

10 nM dox

 

Well 19Well 20Well 21Well 22Well 23Well 24

100 ng HEK293

100 ng hEF1a: mKate

100 ng hEF1a: rtTA

100 ng GmabH

100 ng GmabL

100 ng CD79A-TCS-G4

100 ng CD79B-TCS-G4

100 ng TRE: Syk-TEVp

100 ng UAS: eYFP
100 ng Lyn

10 nM dox

 

100 ngHEK293

100 nghEF1a: mKate

100 nghEF1a: rtTA

50 ng GmabH

50 ng GmabL

50 ng CD79A-TCS-G4

50 ng CD79B-TCS-G4

100 ng TRE: Syk-TEVp

100 ng UAS: eYFP
100 ng Lyn

10 nM dox

 

100 ng HEK293

100 ng hEF1a: mKate

100 ng hEF1a: rtTA

25 ng GmabH

25 ng GmabL

25 ng CD79A-TCS-G4

25 ng CD79B-TCS-G4

100 ngTRE: Syk-TEVp

100 ng UAS: eYFP
100 ng Lyn

10nM dox

 

100 ng HEK293

100 ng hEF1a: mKate

100 ng hEF1a: rtTA

12.5 ng GmabH

12.5 ng GmabL

12.5 ng CD79A-TCS-G4

12.5 ng CD79B-TCS-G4

100 ngTRE: Syk-TEVp

100 ngUAS: eYFP
100 ng Lyn

10 nM dox

 

100 ng HEK293

100 ng hEF1a: mKate

100 ng hEF1a: rtTA

6.25 ng GmabH

6.25 ng GmabL

6.25 ng CD79A-TCS-G4

6.25 ng CD79B-TCS-G4

100 ng TRE: Syk-TEVp

100 ng UAS: eYFP
100 ng Lyn

10 nM dox

 

100 ng HEK293

100 ng hEF1a: mKate

100 ng hEF1a: rtTA

3.125 ng GmabH

3.125 ng GmabL

3.125 ng CD79A-TCS-G4

3.125 ng CD79B-TCS-G4

100 ng TRE: Syk-TEVp

100 ng UAS: eYFP
100 ng Lyn

10 nM dox

 

 

 

 

 

 

Test 2

 

UAS: mKate is TAL14/UAS: mKate

For anti-IgM activation, used 1ug per well (0.5uL of 2mg/mL stock) to get a final concentration of 2ug/mL.  This value was chosen based on a paper where they activated with 10ug antibody/mL (where there were 10^7 B cells per mL).  Given that confluent HEK293 tends to be around 10^6 cells/mL (on the low side) and that there's 500uL media in each well, it seemed reasonable to activate with 1ug.

 

Well 1
Well 2
Well 3
Well 4
Well 5
Well 6

untransfected

(calibration)

eBFP bleed throngh controlleaky mKate control

 

TEVp control

(nonspecific G4 cleavage)

TEVp control

(nonspecific G4 cleavage)

HEK293

 

 HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng hEF1a: Lyn

100 ng GmabH

100 ng GmabL

100 ng CD79A

100 ng CD79B

100 ng Dummy

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

100 ng GmabH

100 ng GmabL

100 ng CD79A

100 ng CD79B

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng UAS: mKate

100 ng hEF1a: Lyn

100 ng GmabH

100 ng GmabL

100 ng CD79A

100 ng CD79B

100 ng TRE: Gal4-VP16

1000 nM dox

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng UAS: mKate

100 ng hEF1a: Lyn

100 ng GmabH

100 ng GmabL

100 ng CD79A-TCS-G4

100 ng CD79B

100 ng Dummy

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng UAS: mKate

100 ng hEF1a: Lyn

100 ng GmabH

100 ng GmabL

100 ng CD79A-TCS-G4

100 ng CD79B

100 ng Dummy

Anti-IgM


Well 7Well 8Well 9Well 10Well 11Well 12

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

100 ng GmabH

100 ng GmabL

100 ng CD79A-TCS-G4

100 ng CD79B

0 nM dox

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

100 ng GmabH

100 ng GmabL

100 ng CD79A-TCS-G4

100 ng CD79B

0 nM dox

Anti-IgM


HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

100 ng GmabH

100 ng GmabL

100 ng CD79A-TCS-G4

100 ng CD79B

1000 nM dox

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

100 ng GmabH

100 ng GmabL

100 ng CD79A-TCS-G4

100 ng CD79B

1000 nM dox

Anti-IgM

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

50 ng GmabH

50 ng GmabL

50 ng CD79A-TCS-G4

50 ng CD79B

200 ng Dummy

1 nM dox

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

50 ng GmabH

50 ng GmabL

50 ng CD79A-TCS-G4

50 ng CD79B

200 ng Dummy

1 nM dox

Anti-IgM

Well 13Well 14Well 15Well 16Well 17Well 18

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

50 ng GmabH

50 ng GmabL

50 ng CD79A-TCS-G4

50 ng CD79B

200 ng Dummy

100 nM dox

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

50 ng GmabH

50 ng GmabL

50 ng CD79A-TCS-G4

50 ng CD79B

200 ng Dummy

100 nM dox

Anti-IgM

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

25 ng GmabH

25 ng GmabL

25 ng CD79A-TCS-G4

25 ng CD79B

300 ng Dummy

10 nM dox

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

25 ng GmabH

25 ng GmabL

25 ng CD79A-TCS-G4

25 ng CD79B

300 ng Dummy

10 nM dox

Anti-IgM


HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

12.5 ng GmabH

12.5 ng GmabL

12.5 ng CD79A-TCS-G4

12.5 ng CD79B

350 ng Dummy

1 nM dox

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

12.5 ng GmabH

12.5 ng GmabL

12.5 ng CD79A-TCS-G4

12.5 ng CD79B

350 ng Dummy

1 nM dox

Anti-IgM

Well 19Well 20Well 21Well 22Well 23Well 24

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

12.5 ng GmabH

12.5 ng GmabL

12.5 ng CD79A-TCS-G4

12.5 ng CD79B

350 ng Dummy

100 nM dox

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

12.5 ng GmabH

12.5 ng GmabL

12.5 ng CD79A-TCS-G4

12.5 ng CD79B

350 ng Dummy

100 nM dox

Anti-IgM

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

6.25 ng GmabH

6.25 ng GmabL

6.25 ng CD79A-TCS-G4

6.25 ng CD79B

375 ng Dummy

0 nM dox

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

6.25 ng GmabH

6.25 ng GmabL

6.25 ng CD79A-TCS-G4

6.25 ng CD79B

375 ng Dummy

0 nM dox

Anti-IgM

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

6.25 ng GmabH

6.25 ng GmabL

6.25 ng CD79A-TCS-G4

6.25 ng CD79B

375 ng Dummy

1000 nM dox

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

6.25 ng GmabH

6.25 ng GmabL

6.25 ng CD79A-TCS-G4

6.25 ng CD79B

375 ng Dummy

1000 nM dox

Anti-IgM

 

 

 

 

 

 

 

 

Well 1
Well 2
Well 3
Well 4
Well 5
Well 6

untransfected

(calibration)

eBFP bleed throngh controlleaky mKate control

TEVp control

(nonspecific G4 cleavage)

TEVp control

(nonspecific G4 cleavage)

TEVp control

(nonspecific G4 cleavage)

 HEK293

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng hEF1a: Lyn

100 ng GmabH

100 ng GmabL

100 ng CD79A

100 ng CD79B

100 ng Dummy

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

100 ng GmabH

100 ng GmabL

100 ng CD79A

100 ng CD79B

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng UAS: mKate

100 ng hEF1a: Lyn

100 ng GmabH

100 ng GmabL

100 ng CD79A

100 ng CD79B

100 ng TRE: Gal4-VP16

1000 nM dox

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng UAS: mKate

100 ng hEF1a: Lyn

100 ng GmabH

100 ng GmabL

100 ng CD79A-TCS-G4

100 ng CD79B

100 ng Dummy

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng UAS: mKate

100 ng hEF1a: Lyn

100 ng GmabH

100 ng GmabL

100 ng CD79A-TCS-G4

100 ng CD79B

100 ng Dummy

Anti-IgM


Well 7Well 8Well 9Well 10Well 11Well 12

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

100 ng GmabH

100 ng GmabL

100 ng CD79A-TCS-G4

100 ng CD79B

0 nM dox

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

100 ng GmabH

100 ng GmabL

100 ng CD79A-TCS-G4

100 ng CD79B

0 nM dox

Anti-IgM


HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

100 ng GmabH

100 ng GmabL

100 ng CD79A-TCS-G4

100 ng CD79B

1000 nM dox

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

100 ng GmabH

100 ng GmabL

100 ng CD79A-TCS-G4

100 ng CD79B

1000 nM dox

Anti-IgM

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

50 ng GmabH

50 ng GmabL

50 ng CD79A-TCS-G4

50 ng CD79B

200 ng Dummy

1 nM dox

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

50 ng GmabH

50 ng GmabL

50 ng CD79A-TCS-G4

50 ng CD79B

200 ng Dummy

1 nM dox

Anti-IgM

Well 13Well 14Well 15Well 16Well 17Well 18

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

50 ng GmabH

50 ng GmabL

50 ng CD79A-TCS-G4

50 ng CD79B

200 ng Dummy

100 nM dox

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

50 ng GmabH

50 ng GmabL

50 ng CD79A-TCS-G4

50 ng CD79B

200 ng Dummy

100 nM dox

Anti-IgM

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

25 ng GmabH

25 ng GmabL

25 ng CD79A-TCS-G4

25 ng CD79B

300 ng Dummy

10 nM dox

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

25 ng GmabH

25 ng GmabL

25 ng CD79A-TCS-G4

25 ng CD79B

300 ng Dummy

10 nM dox

Anti-IgM


HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

12.5 ng GmabH

12.5 ng GmabL

12.5 ng CD79A-TCS-G4

12.5 ng CD79B

350 ng Dummy

1 nM dox

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

12.5 ng GmabH

12.5 ng GmabL

12.5 ng CD79A-TCS-G4

12.5 ng CD79B

350 ng Dummy

1 nM dox

Anti-IgM

Well 19Well 20Well 21Well 22Well 23Well 24

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

12.5 ng GmabH

12.5 ng GmabL

12.5 ng CD79A-TCS-G4

12.5 ng CD79B

350 ng Dummy

100 nM dox

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

12.5 ng GmabH

12.5 ng GmabL

12.5 ng CD79A-TCS-G4

12.5 ng CD79B

350 ng Dummy

100 nM dox

Anti-IgM

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

6.25 ng GmabH

6.25 ng GmabL

6.25 ng CD79A-TCS-G4

6.25 ng CD79B

375 ng Dummy

0 nM dox

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

6.25 ng GmabH

6.25 ng GmabL

6.25 ng CD79A-TCS-G4

6.25 ng CD79B

375 ng Dummy

0 nM dox

Anti-IgM

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

6.25 ng GmabH

6.25 ng GmabL

6.25 ng CD79A-TCS-G4

6.25 ng CD79B

375 ng Dummy

1000 nM dox

 

HEK293

100 ng hEF1a: eBFP

100 ng hEF1a: rtTA

100 ng TRE: Syk-TEVp

100 ng UAS: mKate

100 ng hEF1a: Lyn

6.25 ng GmabH

6.25 ng GmabL

6.25 ng CD79A-TCS-G4

6.25 ng CD79B

375 ng Dummy

1000 nM dox

Anti-IgM

 

 

 

 

 

  • No labels