| 1. |
- Clausson, Carl-Magnus, et al.
(författare)
-
Increasing the dynamic range of in situ PLA
- 2011
-
Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 8:11, s. 892-893
-
Tidskriftsartikel (refereegranskat)
|
|
| 2. |
- Gavrilovic, Milan, et al.
(författare)
-
Automated Classification of Multicolored Rolling Circle Products in Dual-Channel Wide-Field Fluorescence Microscopy
- 2011
-
Ingår i: Cytometry Part A. - : Wiley. - 1552-4922. ; 79A:7, s. 518-527
-
Tidskriftsartikel (refereegranskat)abstract
- Specific single-molecule detection opens new possibilities in genomics and proteomics, and automated image analysis is needed for accurate quantification. This work presents image analysis methods for the detection and classification of single molecules and single-molecule interactions detected using padlock probes or proximity ligation. We use simple, widespread, and cost-efficient wide-field microscopy and increase detection multiplexity by labeling detection events with combinations of fluorescence dyes. The mathematical model presented herein can classify the resulting point-like signals in dual-channel images by spectral angles without discriminating between low and high intensity. We evaluate the methods on experiments with known signal classes and compare to classical classification algorithms based on intensity thresholding. We also demonstrate how the methods can be used as tools to evaluate biochemical protocols by measuring detection probe quality and accuracy. Finally, the method is used to evaluate single-molecule detection events in situ.
|
|
| 3. |
- Leuchowius, Karl Johan, et al.
(författare)
-
In situ proximity ligation assay for microscopy and flow cytometry
- 2011
-
Ingår i: Current Protocols in Cytometry. - : Wiley. - 1934-9297 .- 1934-9300. ; Suppl 56, s. Unit 9.36-
-
Tidskriftsartikel (refereegranskat)abstract
- The ability to study proteins and protein interactions inside cells and tissues is important for elucidating how cells function in health and disease. The in situ proximity ligation assay (in situ PLA) presented here can be used to visualize proteins, protein-protein interactions, and post-translational modifications in cells and tissues. The method is based upon the use of antibodies that target the proteins involved in an interaction; hence, the method has the advantage that it can be used in clinical specimens, providing localized, quantifiable single molecule detection in single cells. This unit describes how in situ PLA can be used with fluorescence microscopy and flow cytometry to study proteins (obtaining high sensitivity and specificity of detection) and protein interactions. It also includes information on expected results and information on how to troubleshoot the assay.
|
|
| 4. |
- Weibrecht, Irene, et al.
(författare)
-
Simultaneous visualization of both signaling cascade activity and end-point gene expression in single cells
- 2011
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:5, s. e20148-
-
Tidskriftsartikel (refereegranskat)abstract
- We have developed an approach for simultaneous detection of individual endogenous protein modifications and mRNA molecules in single cells in situ. For this purpose we combined two methods previously developed in our lab: in situ proximity ligation assay for the detection of individual protein interactions and -modifications and in situ detection of single mRNA molecules using padlock probes. As proof-of-principle, we demonstrated the utility of the method for simultaneous detection of phosphorylated PDGFRβ and DUSP6/MKP-3 mRNA molecules in individual human fibroblasts upon PDGF-BB stimulation. Further we applied drugs disrupting the PDGFRβ signaling pathway at various sites to show that this combined method can concurrently monitor the molecular effect of the drugs, i.e. inhibition of downstream signaling from the targeted node in the signaling pathway. Due to its ability to detect different types of molecules in single cells in situ the method presented here can contribute to a deeper understanding of cell-to-cell variations and can be applied to e.g. pinpoint effector sites of drugs in a signaling pathway.
|
|
| 5. |
- Weibrecht, Irene, 1983-
(författare)
-
Visualizing Interacting Biomolecules In Situ
- 2011
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Intra- and intercellular information is communicated by posttranslational modifications (PTMs) and protein-protein interactions, transducing information over cell membranes and to the nucleus. A cells capability to respond to stimuli by several highly complex and dynamic signaling networks provides the basis for rapid responses and is fundamental for the cellular collaborations required in a multicellular organism. Having received diverse stimuli, being positioned at various stages of the cell cycle or, for the case of cancer, containing altered genetic background, each cell in a population is slightly different from its neighbor. However, bulk analyses of interactions will only reveal an average, but not the true variation within a population. Thus studies of interacting endogenous biomolecules in situ are essential to acquire a comprehensive view of cellular functions and communication. In situ proximity ligation assay (in situ PLA) was developed to investigate individual endogenous protein-protein interactions in fixed cells and tissues and was later applied for detection for PTMs. Progression of signals in a pathway can branch out in different directions and induce expression of different target genes. Hence simultaneous measurement of protein activity and gene expression provides a tool to determine the balance and progression of these signaling events. To obtain this in situ PLA was combined with padlock probes, providing an assay that can interrogate both PTMs and mRNA expression at a single cell level. Thereby different nodes of the signaling pathway as well as drug effects on different types of molecules could be investigated simultaneously. In addition to regulation of gene expression, protein-DNA interactions present a mechanism to manage accessibility of the genomic DNA in an inheritable manner, providing the basis for lineage commitment, via e.g. histone PTMs. To enable analyses of protein-DNA interactions in situ we developed a method that utilizes the proximity dependence of PLA and the sequence selectivity of padlock probes. This thesis presents new methods providing researchers with a set of tools to address cellular functions and communication in complex microenvironments, to improve disease diagnostics and to contribute to hopefully finding cures.
|
|
