Non Invasive Label-Free Studies of Receptor Activation in Lonza® Primary Mesenchymal Stem Cells
 

Non Invasive Label-Free Studies of Receptor Activation in Lonza® Primary Mesenchymal Stem Cells

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There is growing demand for more physiologically-relevant assay platforms, leading to increased adoption of both label-free technologies and stem cells in research. Automated systems are also needed ...

There is growing demand for more physiologically-relevant assay platforms, leading to increased adoption of both label-free technologies and stem cells in research. Automated systems are also needed to facilitate research by increasing cell-based assay efficiency, reproducibility, and performance.

To address these needs, methods utilizing the PerkinElmer EnSpire™ Multimode Plate Reader with Corning® Epic® label-free technology and the JANUS® Automated Workstation have been developed to non-invasively identify and characterize multiple Ion Channel and GPCR activation in Lonza® CloneticsTM Human Umbilical Vein Endothelial Cells (HUVEC) and PoieticsTM human Mesenchymal Stem Cells (hMSC).

By monitoring the ligand-induced dynamic mass redistribution (DMR) in living cells with no need for cellular or ligand modification, the EnSpire label-free platform detects cellular responses from endogenously expressed receptors and ion channels. This obviates the need to engineer cells to over-express receptors of interest, thus greatly reducing the possibility of altering cellular biology.

These data demonstrate the applicability of label-free technology for primary and stem cell research and the utility of liquid handling automation to ensure highly reproducible data from these primary cell assays.

For more information about the EnSpire™ Multimode Plate Reader, please visit http://bit.ly/17fwGwx

For more information about the JANUS® Automated Workstation, please visit http://bit.ly/1cdJpSi

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    Non Invasive Label-Free Studies of Receptor Activation in Lonza® Primary Mesenchymal Stem Cells Non Invasive Label-Free Studies of Receptor Activation in Lonza® Primary Mesenchymal Stem Cells Presentation Transcript

    • ® Lonza Non-Invasive Label-Free Studies of Receptor Activation in Primary & Mesenchymal ® Multimode Plate Reader & JANUS® Automated Workstation Stem Cells Using the EnSpire Heidi Morgan, M.S., Vincent Dupriez, Ph.D., Tim Cloutier, Ph.D., PerkinElmer, Inc. Waltham, MA USA Kristin Atze, Leon de Bruin, Lonza Cologne GmBH, Koeln, Germany ATP M anual Buffer M anual 20 0 20 25 HUVEC (3000 c/w) 40 Isoproterenol Histamine 20 ATP 0 -12 -10 -20 -8 EC50 0.0002087 6.153e-010 Ligand Rim lon e µM ] Response (pm) 25 1 0 -8 -50 -6 -4 [Ligand], M -2 Bithionol Pinacidil -100 -150 Bithionol Pinacidil EC50 2.871e-005 9.240e-005 Label-free robustly monitors the activation of different ion channels in hMSCs. • The absolute response (pm) +/- SD of mean with various ion channel activators and several negative control compounds. • Capsaicin (a known activator of the TRPV1 vanilloid/capsaicin receptor, potentially activating another type of ion channel at the high concentration used here) and Thapsigargin (potent inhibitor of sarco-endoplasmic reticulum Ca2+-ATPases), provide large positive pm responses in hMSC. • Bithionol (an activator of calcium-activated potassium channels) and Pinacidil (an activator of ATP-sensitive potassium channels) provide large negative pm responses. 60 -8 -50 -6 Isoproterenol Histamine ATP -4 [ATP], M Three cell types were used to test the response of either recombinantly or endogenously expressed Purinergic receptors to ATP: Human astrocytoma cells (1321N1), human mesenchymal stem cells (hMSC), and human umbilical vein endothelial cells (HUVEC). A robust 50-fold window (pm) is observed with the endogenously expressed receptors, demonstrating the sensitivity of the EnSpire with label-free platform. 80 2500 c/w 5000 c/w 10000 c/w 15000 c/w 40 20 0 -6 -4 [Ligand], M EC50 9.031e-007 3.729e-007 6.034e-007 7.924e-007 0 -10 -8 -6 -4 -2 6 [Ligand], M -20 EC50 ~ 1.949e-009 2.146e-006 2.143e-006 Isoproterenol Histamine ATP EC50 1.287e-009 2.024e-007 1.438e-006 Label-free ATP Receptor Antagonist Studies in hMSC (2500 c/w) 100 3000 c/w Response (pm) 60 ATP 20 -12 100 1500 c/w 300 Histamine -2 Label-free Histamine Receptor Antagonist Studies in hMSC (2500 c/w) ATP Dose Responses Against Titrating HUVEC Densities ATP Dose Responses Against Titrating hMSC Densities 40 Representative DRCs for several agonists in both cell types: • Histamine and ATP act in a similar manner against the endogenous Histamine and Purinergic receptors in both hMSC and HUVEC (positive DMR response). • Isoproterenol has a differential effect in hMSC relative to the HUVEC system (negative DMR response). • This points to potentially shared (ATP, Histamine) or divergent (isoproterenol) couplings and/or divergent cellular response to a common coupling for the receptors of these ligands. 6000 c/w 200 12000 c/w 100 0 -10 -2 -100 -8 -6 -4 -2 Response (pm) 132N1-rP2Y11 (10000 c/w) hMSC (2500 c/w) HUVEC (3000 c/w) EC50 7.469e-007 7.923e-007 5.949e-007 -4 Clobenpropit 50 Histamine 0 -10 -8 -6 -4 Cell density titrations of hMSC or HUVEC vs. ATP performed to identify the optimal assay signal and window for study. • Lower numbers of cells per well increases the assay window by ~ 3-fold relative to higher densities, thus providing an additional cost savings benefit when running this simple assay workflow. Suramin 0 -10 -8 -6 -4 -2 [Ligand], M -50 -50 12000 c/w 6000 c/w 3000 c/w 1500 c/w ATP 50 [Ligand], M [Ligand], M EC50 7.994e-007 4.168e-007 5.278e-007 7.876e-007 -2 Response (pm) -10 -6 [Ligand], M 0 Response (pm) Label-free Ligand Dose Response Curves on HUVEC (12000 c/w) Response (pm) hM SC (2500 c/w) 50 Corning® and Epic® are registered trademarks of Corning Incorporated. 3-6 reps per bar The label-free platform robustly monitors the activation of GPCRs coupled to various pathways within multiple cell types. • The absolute response (pm) +/- SD of mean with various GPCR activators and several negative control compounds. • AM251, LPI and rimonabant are three known agonists of the GPR55 cannabinoid receptor, which is known to couple only to Gα12/13 proteins. Response (pm) 132N1-rP2Y11 (10000 c/w) 150 15000 c/w 10000 c/w 5000 c/w 2500 c/w [Ligand], M Isoproterenol 200 -20 0 Ligand 3-6 reps per bar 60 250 -8 AM AM 25 1 Ligand 3-6 reps per bar Label-free Ligand Dose Response Curves on hMSC (2500 c/w) ATP Effects On Different Cell Types -10 -2 0 0 Robust Label-free Detection -12 -4 50 Manual 0.71 6% Bu ffe rA EC50 8.941e-007 3.329e-006 -6 Label-Free Response with Ion Channel Ligands in hMSC (2000 c/w) µM ] Automated Z' 0.81 CV, max 3% 40 20 All liquid handling steps were performed both manually and on the JANUS Automated Workstation with comparable results. • The advantage of automating the label-free steps is to provide a robust and reliable easy-to-use protocol resulting in significant time savings and enhanced reproducibility. 3 20 Well Number [Ligand], M ATP (Automated) ATP (Manual) 20 15 10 -8 Capsaicin Thapsigargin µM ] 5 0 60 µM ] 0 -2 Thapsigargin 20 -20 µM ] -4 Capsaicin -10 µM ] -6 40 80 ne [25 Ca 0 ps ac in [25 0 Ca ffe ine Ni [25 flu 0 mi cA cid Th [25 ap 0 sig arg in [40 Pip eri din e[ 25 Bi 0 thi on ol [25 0 Pin ac idi l [2 Iso 50 va gu cin e[ 25 Ga 0 bo xa do l [2 50 -8 [16 µM LP ] I [7 on 0µ ab an M] t [2 Br 00 ad µM yk ini ] His n[ tam 1µ M] ine [50 0µ PG M] E1 [25 0µ PG M] E2 [50 µM S1 ] P[ 5µ AT M] P[ Fo 50 rsk 0µ oli Iso M] n[ pro 50 ter 0µ en M] ol [50 0µ M] LP A[ 50 NE µM CA ] [50 µM Bu ] ffe rA lon e -20 -10 [7 µM LP Rim ] I [8 on 0µ ab M] an Br t [9 ad 0µ yk ini M] n[ His 10 tam 0µ M] ine [10 0µ PG M] E1 [10 0µ PG M] E2 [20 µM S1 ] P[ 2µ AT M] P[ Fo 10 rsk 0µ oli Iso M] n[ pro 10 ter 0µ en M] ol [10 0µ M] LP A[ 20 NE µM CA ] [20 µM Bu ] ffe rA lon e -12 40 100 µM ] 0 40 60 µM ] 20 40 60 120 µM ] 40 Buffer Automated Absolute Response (pm) 60 ATP Automated Absolute Response (pm) ATP (M anual) 60 Absolute Response (pm) ATP (Automated) 80 80 60 hMSC (2000 c/w) 140 HUVEC 12000 c/w 80 Label-Free Response with Ion Channel Ligands in hMSC (2000 c/w) Label-free Ion Channel Studies In hMSC 100 hMSC 2500 c/w 80 100 100 Cell-Based Assays Epic® technology measures changes in light refraction resulting from dynamic mass redistribution (DMR) within the cell which occurs in response to receptor activation or deactivation in a zone within the cell’s monolayer. The change is indicated by a change in wavelength. Label-free Receptor Panning in HUVEC Cultures Response (pm) 100 Label-free Ion Channel Receptor Panning µM ] Comparison of JANUS Automated vs. Manual Assays on hMSC (2500 c/w) 5 Label-free GPCR Receptor Panning Label-free Receptor Panning in hMSC Cultures JANUS Automation vs. Manual Label-free Measurements of ATP Activation in hMSC Response (pm) There is growing demand for more physiologically-relevant assay platforms, leading to increased adoption of both label-free technologies and stem cells in research. Automated systems are also needed to facilitate research by increasing cell-based assay efficiency, reproducibility, and performance. To address these needs, methods utilizing the PerkinElmer EnSpire™ Multimode Plate Reader with Corning® Epic® label-free technology and the JANUS® Automated Workstation have been developed to noninvasively identify and characterize multiple Ion Channel and GPCR activation in Lonza® CloneticsTM Human Umbilical Vein Endothelial Cells (HUVEC) and PoieticsTM human Mesenchymal Stem Cells (hMSC). By monitoring the ligandinduced dynamic mass redistribution (DMR) in living cells with no need for cellular or ligand modification, the EnSpire labelfree platform detects cellular responses from endogenously expressed receptors and ion channels. This obviates the need to engineer cells to over-express receptors of interest, thus greatly reducing the possibility of altering cellular biology. These data demonstrate the applicability of label-free technology for primary and stem cell research and the utility of liquid handling automation to ensure highly reproducible data from these primary cell assays. 4 Automating Stem Cell Assays Do pa mi 2 Abstract Response (pm) 1 Histamine Clobenpropit EC50 2.377e-006 ~ 0.2982 ATP Suramin EC50 1.158e-006 0.01510 Testing the specificity of the agonist response using antagonists in the hMSC Purinergic and Histamine pathways. • Suramin and Clobenpropit clearly inhibit ATP and Histamine agonist activity, respectively. • This inhibition confirms that Purinergic and Histamine receptor activation can be robustly measured using the EnSpire label-free cellular assay. The EnSpire Label-free Platform Offers: • A versatile tool for pathway-independent, global analysis of basic systems biological research and physiologically-relevant drug discovery. • A non-invasive tool to detect GPCR and Ion channel activation in stem and primary cell types. o Suggesting it is a viable platform for studying embryonic stem cells and derived cell types. • In conjunction with the JANUS AWS, significant reduced cost, assay development time and reagents requirements for primary and stem cell research. • In collaboration with the high quality and fully optimized Lonza products, a sensitive and robust system that easily detects both endogenously and recombinantly expressed receptors in receptor panning experiments. PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com