Page History

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title	Trial 1 Transfection Plan

Expand

title	Microscopy Plate

Well 1

B Cells

Well 2

HEK 293

Permeabilized

(Unstained)

Well 3

HEK293

Permeabilized

Well 4

HEK293

+ Dummy (500ng)

+ hEF1a:mKate

(500ng)

Permeabilized

Well 5

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:LilrB2 (500ng)

Permeabilized

Well 6

Well 7

B Cells

Well 8

HEK 293

Permeabilized

(Unstained)

Well 9

HEK293

Permeabilized

Well 10

HEK293

+ Dummy (500ng)

+ hEF1a:mKate

(500ng)

Permeabilized

Well 11

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:LilrB2 (500ng)

Permeabilized

Well 12

Well 13

B Cells

Well 14

HEK 293

Non-Permeabilized

(Unstained)

Well 15

HEK293

Non-Permeabilized

Well 16

HEK293

+ Dummy (500ng)

+ hEF1a:mKate

(500ng)

Non-Permeabilized

Well 17

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:LilrB2 (500ng)

Non-Permeabilized

Well 18

Well 19

B Cells

Well 20

HEK 293

Non-Permeabilized

(Unstained)

Well 21

HEK293

Non-Permeabilized

Well 22

HEK293

+ Dummy (500ng)

+ hEF1a:mKate

(500ng)

Non-Permeabilized

Well 23

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:LilrB2 (500ng)

Non-Permeabilized

Well 24

Expand

title	Cytometry Plate

Well 1

B Cells

Well 2

HEK 293

Well 3

HEK293

Well 4

HEK293

+ Dummy (500ng)

+ hEF1a:mKate (500ng)

Well 5

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:LilrB2 (500ng)

Well 6

Well 7

B Cells

Well 8

HEK 293

Well 9

HEK293

Well 10

HEK293

+ Dummy (500ng)

+ hEF1a:mKate (500ng)

Well 11

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:LilrB2 (500ng)

Well 12

TRIAL 2

Expand

title	Trial 2 Transfection Plan

Well 1 HEK293 (Unstained)	Well 2 HEK293 (Stained)	Well 3 HEK293 hEF1a:LilrB2 (Stained)	Well 4 HEK293 (Unstained)	Well 5 HEK293 (Stained)	Well 6 HEK293 hEF1a:LilrB2 (Stained)
Well 7	Well 8	Well 9	Well 10	Well 11	Well 12
Well 13	Well 14	Well 15	Well 16	Well 17	Well 18
Well 19	Well 20	Well 21	Well 22	Well 23	Well 24

Results:

Expand

title	LilrB2 Trial 1

Microscopy

Permeabilized	Non-permeabilized	Non-permeabilized stained control

Cytometry

Tube 2	Tube 3	Tube 4	Tube 5



Tube 8	Tube 9	Tube 10	Tube 11

Overlay of 4 and 5:

hef1a:mKate + dummy		hef1a:mKate + hef1a:LilrB2
	+		=

...

Expand

title	Trial 1 Transfection Plan

Expand

title	Microscopy Plate

Well 1

B Cells

Well 2

HEK 293

Permeabilized

Well 3

HEK293

+ Dummy

(1000ng)

Permeabilized

Well 4

HEK293

+ Dummy (500ng)

+ hEF1a:mKate

(500ng)

Permeabilized

Well 5

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:PirB (500ng)

Permeabilized

Well 6

Well 7

B Cells

Well 8

HEK 293

Permeabilized

Well 9

HEK293

+ Dummy

(1000ng)

Permeabilized

Well 10

HEK293

+ Dummy (500ng)

+ hEF1a:mKate

(500ng)

Permeabilized

Well 11

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:PirB (500ng)

Permeabilized

Well 12

Well 13

B Cells

Well 14

HEK 293

Non-Permeabilized

Well 15

HEK293

+ Dummy

(1000ng)

Non-Permeabilized

Well 16

HEK293

+ Dummy (500ng)

+ hEF1a:mKate

(500ng)

Non- Permeabilized

Well 17

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:PirB (500ng)

Non-Permeabilized

Well 18

Well 19

B Cells

Well 20

HEK 293

Non-Permeabilized

Well 21

HEK293

+ Dummy

(1000ng)

Non-Permeabilized

Well 22

HEK293

+ Dummy (500ng)

+ hEF1a:mKate

(500ng)

Non- Permeabilized

Well 23

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:PirB (500ng)

Non-Permeabilized

Well 24

Expand

title	Cytometry Plate

Well 1

B Cells

Well 2

HEK 293

Well 3

HEK293

+ Dummy (1000ng)

Well 4

HEK293

+ Dummy (500ng)

+ hEF1a:mKate (500ng)

Well 5

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:PirB (500ng)

Well 6

Well 7

B Cells

Well 8

HEK 293

Well 9

HEK293

+ Dummy (1000ng)

Well 10

HEK293

+ Dummy (500ng)

+ hEF1a:mKate (500ng)

Well 11

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:PirB (500ng)

Well 12

TRIAL 2

Expand

title	Trial 2 Transfection Plan

Well 1 HEK293 (Unstained)	Well 2 HEK293 (Stained)	Well 3 HEK293 hEF1a:PirB (Stained)	Well 4 HEK293 (Unstained)	Well 5 HEK293 (Stained)	Well 6 HEK293 hEF1a:PirB (Stained)
Well 7	Well 8	Well 9	Well 10	Well 11	Well 12
Well 13	Well 14	Well 15	Well 16	Well 17	Well 18
Well 19	Well 20	Well 21	Well 22	Well 23	Well 24

Results:

Expand

title	PirB Trial 1

Microscopy


Control	Permeabilized	Non-permeabilized	LilrB2 Paper

Cytometry

Expand

title	Gating

SSC-A vs FSC-A	FSC-H vs FSC-W	SSC-H vs SSC-W	Yellow vs Red

Tube 1	Tube 2	Tube 3	Tube 4

Tube 5	Tube 6	Tube 7	Tube 8

...

Expand

title	Trial 2

Expand

title	Microscopy Plate

Well 1

B Cells

Well 2

HEK 293

Permeabilized

(Unstained)

Well 3

HEK293

Permeabilized

Well 4

HEK293

+ Dummy (500ng)

+ hEF1a:mKate

(500ng)

Permeabilized

Well 5

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:PirB (500ng)

Permeabilized

Well 6

Well 7

B Cells

Well 8

HEK 293

Permeabilized

(Unstained)

Well 9

HEK293

Permeabilized

Well 10

HEK293

+ Dummy (500ng)

+ hEF1a:mKate

(500ng)

Permeabilized

Well 11

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:PirB (500ng)

Permeabilized

Well 12

Well 13

B Cells

Well 14

HEK 293

Non-Permeabilized

(Unstained)

Well 15

HEK293

Non-Permeabilized

Well 16

HEK293

+ Dummy (500ng)

+ hEF1a:mKate

(500ng)

Non- Permeabilized

Well 17

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:PirB (500ng)

Non-Permeabilized

Well 18

Well 19

B Cells

Well 20

HEK 293

Non-Permeabilized

(Unstained)

Well 21

HEK293

Non-Permeabilized

Well 22

HEK293

+ Dummy (500ng)

+ hEF1a:mKate

(500ng)

Non- Permeabilized

Well 23

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:PirB (500ng)

Non-Permeabilized

Well 24

Expand

title	Cytometry Plate

Well 1

B Cells

Well 2

HEK 293

Well 3

HEK293

Well 4

HEK293

+ Dummy (500ng)

+ hEF1a:mKate (500ng)

Well 5

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:PirB (500ng)

Well 6

Well 7

B Cells

Well 8

HEK 293

Well 9

HEK293

Well 10

HEK293

+ Dummy (500ng)

+ hEF1a:mKate (500ng)

Well 11

HEK293

+ hEF1a:mKate (500ng)

+ hEF1a:PirB (500ng)

Well 12

Analysis:

Cytometry..

LilrB2 and PirB Trial 1: The plots for the mkate only and mkate + receptor cell populations are very similar. This is most probably due to the bleed through of the red channel into the yellow channel (the 'sensitivity' of the PMTs for the yellow channel was increased because the yellow fluorophore was not very bright).

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Versions Compared

Old Version 88

New Version 89

Key

Well 1

Well 2

Well 3

Well 4

Well 5

Well 6

Well 7

Well 8

Well 9

Well 10

Well 11

Well 12

Well 13

Well 14

Well 15

Well 16

Well 17

Well 18

Well 19

Well 20

Well 21

Well 22

Well 23

Well 24

Well 1

Well 2

Well 3

Well 4

Well 5

Well 6

Well 7

Well 8

Well 9

Well 10

Well 11

Well 12

Well 1

Well 2

Well 3

Well 4

Well 5

Well 6

Well 7

Well 8

Well 9

Well 10

Well 11

Well 12

Well 13

Well 14

Well 15

Well 16

Well 17

Well 18

Well 19

Well 20

Well 21

Well 22

Well 23

Well 24

Results:

Well 1

Well 2

Well 3

Well 4

Well 5

Well 6

Well 7

Well 8

Well 9

Well 10

Well 11

Well 12

Well 13