Description: 

In this experiment, we are aiming to test whether or not our receptor proteins localize to the cell membrane. To do that, we will have HEK293 cells express LilrB2 and PirB. We will test for membrane localization of the receptors through immunofluorescence. We will use primary antibodies that will bind to LilrB2 and and PirB and secondary antibodies conjugated to fluorophores that will bind to the primary antibodies. 

Cloning

COMPLETE

Description:

Making pEXPRs hEF1a:LilrB2 and hEF1a:PirB.

Plan:
  • Order LilrB2 and PirB gBlocks 
  • Simulate Golden Gate on Geneious and make sure it works 
  • Golden Gate gBlocks into ggDONR
  • Transform bacteria with Golden Gate product and plate 
  • Pick colonies and grow in liquid culture
  • Miniprep cells and verify product 
  • Make glycerol stocks
  • LR into pDEST
  • Transform, pick colonies and grow in liquid culture
  • Miniprep cells and verify product
  • Make glycerol stocks 
  • Midiprep product

 

 

 

Experiment

Protocols:

Immunostaining for flow cytometryImmunostaining for microscopy

Antibodies:

Antibody
Type
Conjugation
Dilution Used
Link
Anti-LilrB2Mouse monoclonal 

Cytometry: 1/2000

Immunofluorescence: 1/2000

http://www.abcam.com/lilrb2-antibody-ab172538.html
Anti-PirBGoat polyclonal 

Cytometry: 1/2000

Immunofluorescence: 1/2000

http://www.scbt.com/datasheet-9609-Pirb-c-19-antibody.html

Donkey Anti-Goat IgG

 Alexa Fluor 488

Cytometry: 1/2000

Immunofluorescence: 1/200 - 1/1000.

 
Donkey Anti-Mouse IgG 

Alexa Fluor 488

Cytometry: 1/2000

Immunofluorescence: 1/200 - 1/1000.

 

Transfection Plans:

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
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

 


Blind Transfection

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
Fixed and stained

 

Well 1

HEK293

(Unstained)

Well 2

HEK293

(Stained)

Well 3

HEK293

hEF1a:eYFP 500ng

hEF1a:LilrB2 500ng

(Stained)

Well 4

HEK293

(Unstained)

Well 5

HEK293

(Stained)

Well 6

HEK293

hEF1a:eYFP 500ng

hEF1a:LilrB2 500ng

(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

Blind Transfection

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
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
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

 


Blind Transfection

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
In this experiment, we're transfecting the receptor fused to YFP to try get some conclusive results as to whether or not the receptor is localizing to the membrane (since the immunostaining didn't give us any results - or rather gave us weird results)

Well 3: control for bleed through

Well 1

HEK293

 

Well 2

HEK293

hEF1a:mKate

Dummy

Well 3

HEK293

hEF1a:PirB-YFP

Dummy

Well 4

HEK293

hEF1a: mKate

hEF1a:PirB-YFP

Well 5

 

Well 6

 

Well 7

HEK293

 

Well 8

HEK293

hEF1a:mKate

Dummy

Well 9

HEK293

hEF1a:PirB-YFP

Dummy

Well 10

HEK293

hEF1a: mKate

hEF1a:PirB-YFP

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:

LilrB2

Microscopy

PermeabilizedNon-permeabilizedNon-permeabilized stained control 
 


Cytometry

Tube 2Tube 3Tube 4Tube 5
  
Tube 8Tube 9Tube 10Tube 11

Overlay of 4 and 5:

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

 

 

Well 1

HEK293

(Unstained)

Well 2

HEK293

(Stained)

Well 3

HEK293

hEF1a:eYFP 500ng

hEF1a:LilrB2 500ng

(Stained)

Well 4

HEK293

(Unstained)

Well 5

HEK293

(Stained)

Well 6

HEK293

hEF1a:eYFP 500ng

hEF1a:LilrB2 500ng

(Stained)

    
UntransfectedTransfected

 

 

Well 1

HEK293

(Unstained)

Well 2

HEK293

(Stained)

Well 3

HEK293

hEF1a:LilrB2

(Stained)

Not gated

Non-fluorescent population gated out

Mean: 326

 

non-fluorescent population gated out

Mean: 377

 

Duplicates

Not gated

Non-fluorescent population gated out

Mean: 280

Non-fluorescent population gated out

Mean: 358

PirB

Microscopy

ControlPermeabilizedNon-permeabilizedLilrB2 Paper

Cytometry

SSC-A vs FSC-AFSC-H vs FSC-WSSC-H vs SSC-WYellow vs Red
Tube 1Tube 2Tube 3Tube 4
Tube 5Tube 6Tube 7Tube 8

Well 4 - mKate bleeds into the yellow channel.. a lot.

Red channelYellow channelOverlay

 

 

Negative ControlPirB-YFP only (no transfection marker) 
 

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).

Next steps:

  • We are going to transfect the receptors without using a transfection marker (Trial 2).

Microscopy..

   Immunostaining 

LilrB2 and PirB Trial 1: For both the receptors, there doesn't seem to be any clear localization of the receptors to the membrane. From the punctate appearance of the cells under the confocal, they seem to be significant localization of the receptors in some subcellular cytoplasmic structure. They may be making it to the membrane; however we are unable to discern that just from the images. Interestingly, this cytoplasmic localization seems to be apparent in the non-permeabilized cells which may mean that the fixing protocol is causing some kind of permeabilization in the cells. It may be of significance to note that in the LilrB2 paper (the one we are trying to replicate), they expressed the receptors under CMV (not hEF1a). Depending on the relative strengths of the promoters, the differing levels of expression (probably higher expression through hEF1a) might be causing the staining pattern that we are seeing.

   YFP fusion 

Evidence is not super convincing that the yellow that we are seeing is not just autofluorescence given that we see something very similar in our negative control. It is likely that PirB is not getting expressed at all, or that the fusion is disturbing it's structure somehow making it not localize to the membrane (the happier scenario). mKate bleedthrough into the yellow channel was an issue. Ideally, we would make an eBFP fusion but considering how hard it was to make the fusions in the first place, that isn't really a feasible solution, we can possibly look into using an IR protein as a transfection marker (Trial 4). Also, it is probably a good idea to DAPI counterstain next time. Zstacks with nuclear staining will help in determining membrane localization (or lack thereof).

Next steps:

  • Is the receptor even getting expressed? Maybe if we transfect TRE:receptor-YFP and run a dox ladder, we can conclude on whether or not there is expression based on whether or not there are varying levels of yellow corresponding to the dox ladder.
  • Run PirB Trial 3 again but with an IR fluorescent transfection marker to try and eliminate bleedthrough (Trial 4)