Genetic Engineering & Biotechnology News

NOV1 2018

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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The Power of Duolink ® fl owPLA Technology Flow Cytometry Detection Kits for Studying Protein-Protein Interactions, Post-Translational Modifi cations, and Low-Abundance Protein Targets Authors: Tracy Adair-Kirk, Jacqueline Day, and Holly Johnston of MilliporeSigma PREPARATION Duolink ® PLA ANALYSIS Step 1. Fix cells, permeabilize, and block as bulk suspension Step 4. Wash and add the PLUS and MINUS PLA probes Step 8. Run flow cytometry instrument Step 9. Data analysis Step 5. Wash and add the ligation solution Step 6. Wash and add the amplification solution Step 7. Wash and add the detection solution Step 2. Aliquot cells in tubes or 96-well plates (+/- filters) Step 3. Add primary antibodies Figure 1 shows the basic steps to perform a Duolink ® assay for fl ow cytometry using a Duolink ® fl owPLA Detection Kit. During the fi rst three steps, the cells are prepared for the Duolink ® assay. Single-cell suspensions are pre-treated with respect to fi xation and permeabilization and then blocked in bulk solution (Step 1). Cells are then aliquoted in tubes or 96-well plates (Step 2) and incubated with primary antibodies (Step 3). The Duolink ® PLA portion of the procedure includes the incubation with the PLUS and MINUS PLA probes (Step 4), ligation (Step 5), amplifi cation (Step 6), and fi nally, incubation with detection solution (Step 7) with washing between each step. Once the Duolink ® PLA procedure is complete, the amount of PLA signal in individual cells of a cell population is measured by fl ow cytometry (Step 8) and the data analyzed (Step 9). The Duolink ® fl owPLA Protocol is Agnostic of Flow Cytometry Instrumentation The same Duolink ® assay performed using the Duolink ® fl owPLA Detection Kit – FarRed to detect a protein-protein interaction was analyzed using multiple fl ow cytometers. Data in Figure 2 show results from the Miltenyi Biotec MACSQuant ® and the Amnis IMAGEStreamX ® instruments, but comparable results were also achieved using the Guava ® easyCyte 8HT and the BD FACSAria™ systems. Figure 2. Duolink ® PLA analyzed using multiple fl ow cytometers produced comparable results. Results show typical histogram from multiple fl ow cytometers illustrating reproducibility and fl exibility of the protocol with respect to instrumentation. A protein-protein interaction was queried via the Duolink ® PLA fl ow cytometry kit with read-out on both the MACSQuant ® with FlowJo ® analysis (A) and the ImageStreamX ® (B). The Cy5 fi lter set was used on both instruments. Note: Some background staining was observed in the negative control compared to unstained cells, but the PLA signal intensity was signifi cantly greater in the experimental samples that had undergone a Duolink ® proximity ligation assay. Duolink ® PLA technology has been used by researchers to study protein events via microscopy and more recently in fl ow cytometry experiments. This article describes the new possibilities that are available to fl ow cytometry users to study protein-protein interactions, post-translational modifi cations, and low- abundance targets. References: For EZH2/H3K27me3: Yoo K and Hennighausen L. EZH2 Methyltransferase and H3K27 Methylation in Breast Cancer. International Journal of Biological Sciences, 8, 59-65 (2011). Au SL, Wong CC, Lee JM, Wong CM, and Ng IO. EZH2-Mediated H3K27me3 is Involved in Epigenetic Repression of Deleted in Liver Cancer 1 in Human Cancers. PLoS One, 8(6), e68226 (2013). Li J, Hart RP, Mallimo EM, Swerdel MR, Kusnecov AW, and Herrup K. EZH2- meidated H3K27 trimethylation mediates neurodegeneration in ataxia-telangiesctasia. Nature Neuroscience, 16, 1745-1753 (2013). Holm K, Grabau D, Lövgren K, Aradottir S, Gruvberger-Saal S, Howlin J, Saal LH, Ethier SP, Bendahl PO, Stål O, Malmström P, Fernö M, Rydén L, Hegardt C, Borg A, and Ringnér M. Global H3K27 trimethylation and EZH2 abundance in breast tumor subtypes. Molecular Oncology, 6(5), 494-506 (2012). For PLA and fl owPLA: Söderberg O, Gullberg M, Jarvius M , Ridderstråle K, Leuchowius KJ, Jarvius J, Wester K, Hydbring P, Bahram F, Larsson LG, and Landegren U. Direct observation of individual endogenous protein complexes PLA by proximity ligation. Nat Methods, 3, 995- 1000 (2006). Leuchowius K, Weibrecht I, Landegren U, Gedda L, and Söderberg O. Flow Cytometric In Situ Proximity Ligation Analyses of Protein Interactions and Post-translational Modifi cation of the Epidermal Growth Factor Receptor Family. Cytometry, Part A, 75, 833- 839 (2009). Avin A, Levy M, Porat Z, and Abramson J. Quantitative analysis of protein- protein interactions and post-translational modifi cation in rare immune populations. Nature Communications, 8:1524, 1-10 (2017). Andersen SS, Hvid M, Pedersen FS, and Deleuran B. Proximity ligation assay combined with fl ow cytometry is a powerful tool for the detection of cytokine receptor dimerization. Cytokine, 64, 54-57 (2013). © 2018 Merck KGaA, Darmstadt, Germany and/or its affi liates. All Rights Reserved. MilliporeSigma and the vibrant M are trademarks of Merck KGaA, Darmstadt, Germany or its affi liates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources. A B Key Sample ID • PLA with O/N amplifi cation • PLA with 4 hr amplifi cation • PLA with 100 min amplifi cation • No primary antibodies control, O/N amplifi cation • Unstained cells Figure 1 Figure 2

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