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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

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