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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.Testing the tango system using a Lyn-TEV cofactor

This will To test whether or not the BCR complex will reach the cell membrane in HEK293 cellsthe signal to noise ratio of the Lyn-TEV tango system.

pEXPR hEF1a: Gmab LightpEXPR

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

PLATE 2

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

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