Membrane Localization of BCR

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

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

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.

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

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. B-Cells should act as a positive control for this experiment.

calibrate cytometer with wells2/8

then run remaining tubes sequentially

image well 1 b-cells alt 7

image well 3 stained HEK293 cells with no DNA alt 9

image well 4 stained HEK293 cells withmkate + Dummy DNA alt 10

image well 5 stained HEK293 with BCR alt 11

image well 13 permeablized b-cells alt 19

image well 15 permeablized HEK293 with no DNA alt 21

image well 16 permeablized HEK293 with mkate+dummy alt 22

image well 17 permeablized HEK293 with BCR alt 23

Although there was a lot of bleedthrough in this experiment, there is a qualitative difference between the control group (with only mKate) and the group also expressing the receptor - while the bleedthrough in the control group forms a tight line, the experimental group has a population of cells with yellow fluorescence levels above what we would expect from this. This increased yellow fluorescence is most likely due to staining of the receptor, as desired (notably, it is in cells with high levels of mKate, which is consistent with high levels of the transfection marker correlating with high levels of our receptor).

Image - experimental group (sample 5) in green, with control group (sample 4) overlaid in black

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

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. B-Cells should act as a positive control for this experiment.

calibrate cytometer with wells2/8

then run remaining tubes sequentially

image well 1 b-cells alt 7

image well 3 stained HEK293 cells with no DNA alt 9

image well 4 stained HEK293 cells withmkate + Dummy DNA alt 10

image well 5 stained HEK293 with BCR alt 11

image well 13 permeablized b-cells alt 19

image well 15 permeablized HEK293 with no DNA alt 21

image well 16 permeablized HEK293 with mkate+dummy alt 22

image well 17 permeablized HEK293 with BCR alt 23

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