- Created by Unknown User (clrichar@mit.edu) on Aug 08, 2014 15:53
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Use anti IgM with a fluorescent fused secondary antibody to test for membrane localization of the BCR complex. Use B-Cells as a control.
This will test whether or not the BCR complex will reach the cell membrane in HEK293 cells.
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
PLATE 1
Well 1 EMPTY | Well 2 HEK293 | Well 3 HEK293 Dummy DNA 1000 ng | Well 4 HEK293 mKate 200 ng Dummy DNA 800 ng | Well 5 HEK293 Gmab L 200 ng Gmab H 200 ng CD79A 200 ng CD79B 200 ng mKate 200 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 10 HEK293 mKate 200 ng Dummy DNA 800 ng | Well 11 HEK293 Gmab L 200 ng Gmab H 200 ng CD79A 200 ng CD79B 200 ng mKate 200 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 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 18 HEK293 Gmab L 200 ng Gmab H 200 ng CD79A 100 ng CD79B 100 ng mKate 200 ng Dummy DNA 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 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 1 EMPTY | Well 2 HEK293 | Well 3 HEK293 Dummy DNA 1000 ng | Well 4 HEK293 mKate 200 ng Dummy DNA 800 ng | Well 5 HEK293 Gmab L 200 ng Gmab H 200 ng CD79A 200 ng CD79B 200 ng mKate 200 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 10 HEK293 mKate 200 ng Dummy DNA 800 ng | Well 11 HEK293 Gmab L 200 ng Gmab H 200 ng CD79A 200 ng CD79B 200 ng mKate 200 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 |
EMPTY | EMPTY | EMPTY | EMPTY | EMPTY | EMPTY |
| EMPTY | EMPTY | EMPTY | EMPTY | EMPTY | EMPTY |
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.
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.
Microscopy Data:
Left: HEK 293 transfected with dummy DNA
Right: HEK 293 transfected with full BCR plasmids
Cytometry data were gated to remove non-cell data points.
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.
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