PBLs were cytospun onto poly-L-lysine (Sigma-Aldrich)-treated coverslips for 5 min at 500 × g to optimize cell attachment and cytoplasmic spread. DCs were seeded onto collagen-treated coverslips to minimize autofluorescence. Imaging was performed using a Zen 2012 LSM700 and LSM780 confocal microscopes (Zeiss) with a Plan-Apochromat 63×/NA 1.4 objective. Duolink proximity ligation assay (PLA) was performed according to manufacturer’s instructions (Olink Biosciences).
Duolink proximity ligation assay pla
Manufactured by Olink
Sourced in Sweden
Duolink proximity ligation assay (PLA) is a laboratory equipment product that enables the detection and analysis of protein-protein interactions. It is a versatile tool that can be used in various applications, including cell biology, molecular biology, and proteomics research.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780, by Zeiss (2 mentions)
Plan apochromat 63 1.4 na objective, by Zeiss (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Mab197, by R&D Systems (1 mentions)
Duolink image tool software, by Olink (1 mentions)
Af 310 na, by R&D Systems (1 mentions)
Mab172, by R&D Systems (1 mentions)
5 protocols using duolink proximity ligation assay pla
1
Cell Attachment and Imaging Optimization
2
CCR7 Oligomerization Detected by PLA
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CCR7 homo-oligomer formation was examined by the Duolink proximity ligation assay (PLA; Olink Bioscience), in which oligomerized CCR7 appears as orange dots representing the presence of a protein-protein interaction complex. Briefly, H9 cells in culture media containing 0.1% BSA were treated with or without CXCL12 or recombinant gp120 in fibronectin-coated wells for 30 min, fixed with 4% paraformaldehyde, and stained with a 7.5 μg/ml anti-human CCR7 mAb (R&D, MAB197) independently conjugated with the complementary oligonucleotide probe generated by Duolink II Probemaker PLUS or MINUS (Olink Bioscience). CCR7/CXCR4 hetero-oligomerization was examined by using anti-human CCR7 and anti-human CXCR4 mAbs (eBioscience, clone 12G5), which were labeled with the PLA-Minus and the PLA-Plus probes, respectively. As a control, anti-human CCR1 mAb (R&D, clone 53504) was used. The complementary DNA strands in close proximity (<40 nm) were amplified using fluorescence, which were visualized using a confocal laser microscope. The images were analyzed using the Duolink Image Tool software (Olink Bioscience).
3
Chemokine Receptor Interactions in BMDC
4
Investigating PKD1-HSP20 Interaction using PLA
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PKD1 association with HSP20 was investigated further by using the Duolink™ Proximity Ligation Assay (PLA) (Olink Bioscience, Uppsala, Sweden). After fixation, permeabilisation and blocking, slides were incubated with primary antibodies against PKD1 and HSP20 raised in two different species. Then PLA probes, which are conjugated with oligonucleotides, were introduced to recognise the primary antibodies. A solution that promotes hybridization between the PLA oligos was then added, where a hybridisation reaction only occurred if the two proteins were in close proximity (<40 nm), but not if they were far apart. This reaction was followed by ligation of the oligonucleotides and a rolling circle amplification (RCA) reaction, where a repeated sequence product was made. This product was then detected using fluorescently labeled oligonucleotides, where a Hsp20-PKD1association appeared as discrete red dots under the microscope. Cells were counterstained with α-actinin to show sarcomeric stained. Slides were finally mounted under coverslips with DUOLINK mounting media and visualised. In order to detect all PLA signals, a series of Z-stack images was collected.
5
In Situ Proximity Ligation Assay
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In situ PLA was performed according to the Duolink® Proximity Ligation Assay PLA (Olink biosciences) protocol with modifications (seeSupplementary Material for details).
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In situ PLA was performed according to the Duolink® Proximity Ligation Assay PLA (Olink biosciences) protocol with modifications (see
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