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.
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Status:
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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
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Protocols: Immunostaining for flow cytometry
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LilrB2
TRIAL 1
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| title | Trial 1 Transfection Plan |
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Permeabilized
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Permeabilized
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Permeabilized
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Permeabilized
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Well 13
B Cells
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Well 14
HEK 293
Non-Permeabilized
(Unstained)
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Well 15
HEK293
Non-Permeabilized
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Well 16
HEK293
+ Dummy (500ng)
(500ng)
Non-Permeabilized
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Well 17
HEK293
+ hEF1a:mKate (500ng)
+ hEF1a:LilrB2 (500ng)
Non-Permeabilized
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Well 18
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Well 19
B Cells
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Well 20
HEK 293
Non-Permeabilized
(Unstained)
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Well 21
HEK293
Non-Permeabilized
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Well 22
HEK293
+ Dummy (500ng)
(500ng)
Non-Permeabilized
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Well 23
HEK293
+ hEF1a:mKate (500ng)
+ hEF1a:LilrB2 (500ng)
Non-Permeabilized
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Well 24
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| title | Cytometry Plate |
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Well 1
B Cells
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Well 2
HEK 293
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Well 3
HEK293
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Well 4
HEK293
+ Dummy (500ng)
+ hEF1a:mKate (500ng)
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Well 5
HEK293
+ hEF1a:mKate (500ng)
+ hEF1a:LilrB2 (500ng)
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Well 6
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Well 7
B Cells
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Well 8
HEK 293
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Well 9
HEK293
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Well 10
HEK293
+ Dummy (500ng)
+ hEF1a:mKate (500ng)
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Well 11
HEK293
+ hEF1a:mKate (500ng)
+ hEF1a:LilrB2 (500ng)
Well 12
TRIAL 2
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Cytometry
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Overlay of 4 and 5:
| hef1a:mKate + dummy | hef1a:mKate + hef1a:LilrB2 | |||
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PirB
TRIAL 1
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| title | Microscopy Plate |
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Well 1
B Cells
Well 2
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Non-Permeabilized
Well 24
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Well 6
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+ Dummy (1000ng)
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Well 10
HEK293
+ Dummy (500ng)
+ hEF1a:mKate (500ng)
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Well 11
HEK293
+ hEF1a:mKate (500ng)
+ hEF1a:PirB (500ng)
Well 12
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Well 1
HEK293
(Unstained)
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Well 2
HEK293
(Stained)
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Well 3
HEK293
hEF1a:PirB
(Stained)
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Well 4
HEK293
(Unstained)
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Well 5
HEK293
(Stained)
Well 6
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Well 8
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Well 9
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Well 10
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Well 11
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Well 12
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Well 13
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Well 14
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Well 15
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Well 16
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Well 17
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Well 18
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Well 19
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Well 20
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Well 21
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Well 22
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Well 23
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Well 24
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Results:
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Microscopy
Cytometry
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TRIAL 2
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| title | Trial 2 |
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| title | Cytometry Plate |
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Results:
LilrB2
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Microscopy
Cytometry
Overlay of 4 and 5:
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PirB
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Microscopy
Cytometry
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Well 4 - mKate bleeds into the yellow channel.. a lot.
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Well 1
B Cells
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Well 2
HEK 293
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Well 3
HEK293
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Well 4
HEK293
+ Dummy (500ng)
+ hEF1a:mKate (500ng)
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Well 5
HEK293
+ hEF1a:mKate (500ng)
+ hEF1a:PirB (500ng)
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Well 6
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Well 7
B Cells
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Well 8
HEK 293
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Well 9
HEK293
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Well 10
HEK293
+ Dummy (500ng)
+ hEF1a:mKate (500ng)
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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).
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.
Next steps:
- Perhaps staining live cells (to skip the fixation step) and hopefully conclude whether or not some of the receptor is making it to the membrane. (We are also going to do the beta amyloid binding experiment despite being unsure whether or not the receptor is making it to the membrane in hopes that it does bind and we can conclude that it is localizing.)
- Once we get the receptor-fluorescent protein fusions out of cloning, we can express them and look for localization. This will tell us whether or not the staining pattern that we're seeing is due to the protein expression or the staining.
- Clone the receptors into a different promoter: CMV or a TRE to determine if high expression levels are the problem. Having inducible expression of the receptors may be helpful later on in determining what the optimal level of receptor expression for the system.
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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)