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Expand
titleTest 1
Expand
titleDescription

Use anti IgM with a fluorescent fused secondary antibody to test for membrane localization of the BCR complex. Use B-Cells as a control. 

Expand
titlePurpose

This will test whether or not the BCR complex will reach the cell membrane in HEK293 cells. 

PLATE 1

Well 5 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 200 ng

CD79B 200 ng

mKate 200 ng

Well 10 

HEK293

mKate 200 ng

Dummy DNA 800 ng

Well 12 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 100 ng

CD79B 100 ng

mKate 200 ng

Dummy DNA 200 ng

Well 18 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 100 ng

CD79B 100 ng

mKate 200 ng

Dummy DNA 200 ng

Well 24 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 100 ng

CD79B 100 ng

mKate 200 ng

Dummy DNA 200 ng

PLATE 2

Well 5 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 200 ng

CD79B 200 ng

mKate 200 ng

Well 10 

HEK293

mKate 200 ng

Dummy DNA 800 ng

Well 12 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 100 ng

CD79B 100 ng

mKate 200 ng

Dummy DNA 200 ng

Expand
titleParts Needed

pEXPR hEF1a: Gmab Light

pEXPR hEF1a: Gmab Heavy

pEXPR hEF1a: CD79A

pEXPR hEF1a: CD79B

ALSO NEED:

Anti IgM primary antibody fused to a yellow alexa dye

B cells

Expand
titleSetup

Well 1

EMPTY

Well 2

HEK293

Well 3

HEK293

Dummy DNA 1000 ng

Well 4

HEK293

mKate 200 ng

Dummy DNA 800 ng

Well 6 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 100 ng

CD79B 100 ng

mKate 200 ng

Dummy DNA 200 ng

Well 7

EMPTY

Well 8 

HEK293

Well 9 

HEK293

Dummy DNA 1000 ng

 

Well 11 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 200 ng

CD79B 200 ng

mKate 200 ng

 

Well 13 

EMPTY

Well 14 

HEK293

Well 15 

HEK293

Dummy DNA 1000 ng

 

Well 16 

HEK293

mKate 200 ng

Dummy DNA 800 ng

 

Well 17 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 200 ng

CD79B 200 ng

mKate 200 ng

 

Well 19 

EMPTY

Well 20

HEK293

Well 21 

HEK293

Dummy DNA 1000 ng

 

Well 22 

HEK293

mKate 200 ng

Dummy DNA 800 ng

 

Well 23 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 200 ng

CD79B 200 ng

mKate 200 ng

 

Well 1

EMPTY

Well 2

HEK293

Well 3

HEK293

Dummy DNA 1000 ng

Well 4

HEK293

mKate 200 ng

Dummy DNA 800 ng

Well 6 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 100 ng

CD79B 100 ng

mKate 200 ng

Dummy DNA 200 ng

Well 7

EMPTY

Well 8 

HEK293

Well 9 

HEK293

Dummy DNA 1000 ng

 

Well 11 

HEK293

Gmab L 200 ng

Gmab H 200 ng

CD79A 200 ng

CD79B 200 ng

mKate 200 ng

 

EMPTY

EMPTY

EMPTYEMPTYEMPTYEMPTY
EMPTYEMPTYEMPTYEMPTYEMPTYEMPTY
Expand
titleExpected Results

We expect to see clear yellow fluorescence around the blue stained nuclei indicating BCR localization to the HEK cell membrane. Moreover Transfection efficiency as measured by our mKate transfection marker should correlate to anti IgM fluorescence in the flow cytometer.

Expand
titleProtocol

Day 1

Cells were seeded using the Gelatin Treatment for Glass Plates protocol (1 mL/well was used) for plate 1. For plate 2 the standard Splitting Cells & seeding plates protocol was used.

Day 2

All cells were transfected based on the above plate map using the Transfection (Lipofectamine 2000) protocol.

Day 3

Cells were left to grow

Day 4

Plate two was run through the flow cytometer. They were prepared using the Immunostaining for Flow Cytometry protocol.

Plate one was used for fluorescent microscopy. It was prepared with the Immunostaining for Fluorescent microscopy protocol. Wells 1-12 were permeablized with Triton-X100 while wells 13-24 were left with their membranes intact.

Expand
titleMicroscopy Data

Microscopy Data:

Left: HEK 293 transfected with dummy DNA

Right: HEK 293 transfected with full BCR plasmids

Image RemovedImage Removed

Expand
titleCytometry Data

Cytometry data were gated to remove non-cell data points.

 

Expand
titleNegative/negative control

Image Removed

Expand
titleUntransfected HEK 293

Image RemovedImage Removed

Expand
titleTransfected w/ Dummy DNA

Image RemovedImage Removed

Expand
titleTransfected w/ mKate only

Image RemovedImage Removed

Expand
titleTransfected w/ BCR Plasmids

Image RemovedImage Removed

 

 

 

 

 

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

Expand
titleExpected Results

 

Expand
titleProtocol

 

Expand
titleMicroscopy Data

 

Expand
titleCytometry Data

 

Expand
titleAnalysis

 

 

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

 

 

 

Expand
titleTest 3
Expand
titleDescription

 

Expand
titlePurpose

 

Expand
titleParts Needed

 

 

 

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
Expand
titleExpected Results

 

Expand
titleProtocol

 

Expand
titleMicroscopy Data

 

Expand
titleCytometry Data

 

Expand
titleAnalysis

 

Expand
titleAnalysis

B-Cells of any kind were unavailable for use as a positive control for this experiment. Moreover the mKate transfection marker in our control did not show as much red fluorescence as was expected. The cytometry data remains inconclusive but the BCR plots suggest that there was BCR localization. Moreover the microscopy clearly shows membrane localization of the BCR in both permeablized and non-permeablized cells. This experiment will be repeated once B-Cells Become available. Additionally in this experiment no untransfected unstained control was planned into the plate and thus we had to collect one on very short notice. For the next experiment this control will be planned into the experiment. A final problem with this experiment was a mix up in sample labels that resulted in a mislabeling of cytometry samples. The data however was sufficiently different for the samples to be re sorted into their original labels. Due to the various problems and limitations in this initial experiment there will be a repeat of this experiment done as soon as B-Cells become available as a positive control.