Membrane Localization of BCR

Test 1

Description

Use anti IgM with a fluorescent fused secondary antibody to test for membrane localization of the BCR complex. Use B-Cells as a control.

Purpose

This will test whether or not the BCR complex will reach the cell membrane in HEK293 cells.

Parts 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

Setup

PLATE 1

Well 1

B-Cells

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

B-Cells

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

B-Cells

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

B-Cells

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 B-Cells	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 B-Cells	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

Expected 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.

Protocol

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

Microscopy Data:

Left: HEK 293 transfected with dummy DNA

Right: HEK 293 transfected with full BCR plasmids