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titleTest 1
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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. 

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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

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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

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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
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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.

 

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titleSetup

 

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titleExpected Results

 

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titleProtocol

 

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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.

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titleMicroscopy Data

Microscopy Data:

Left: HEK 293 transfected with dummy DNA

Right: HEK 293 transfected with full BCR plasmids

Image RemovedImage Removed 

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titleCytometry Data

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

 

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title
Negative/negative control

Image Removed

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titleUntransfected HEK 293

Image RemovedImage Removed

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titleTransfected w/ Dummy DNA

Image RemovedImage Removed

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titleTransfected w/ mKate only

Image RemovedImage Removed

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titleTransfected w/ BCR Plasmids

Image RemovedImage Removed

 

 

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titleAnalysis
Analysis

 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.