Optimization and calibration of 384-well kinetic Ca2+ mobilization assays for the human transient receptor potential cation channels TRPM8, TRPV1, and TRPA1
Development, optimization, and calibration of human transient receptor potential (TRP) channel Ca2+ mobilization assays for TRPM8, TRPV1, and TRPA1 are described. Heterologous expression of hTRPM8 in HEK293T cells was required for anti-TRPM8 antibody staining and TRPM8 agonist induced Ca2+ mobilizat...
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Elsevier
2025-01-01
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| author | David A. Close V. Blair Journigan Paul A. Johnston |
| author_facet | David A. Close V. Blair Journigan Paul A. Johnston |
| author_sort | David A. Close |
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| description | Development, optimization, and calibration of human transient receptor potential (TRP) channel Ca2+ mobilization assays for TRPM8, TRPV1, and TRPA1 are described. Heterologous expression of hTRPM8 in HEK293T cells was required for anti-TRPM8 antibody staining and TRPM8 agonist induced Ca2+ mobilization signals which were both used to optimize transfection efficiency. FLIPR Calcium 6 dye concentration, loading time, and TRPM8 transfected cell seeding density were optimized and a DMSO tolerance of ≤0.2 % was set. The resting baseline relative fluorescent unit (RFUs) signals of the TRPM8 Ca2+ mobilization assay exhibited substantial well-to-well variability, even though such differences were small relative to maximal agonist induced responses. Maximum RFU, cumulative RFU sum, or area under the curve values were extracted from Ca2+ mobilization kinetic data to plot curves and calculate EC50 and IC50 values. Fold over baseline (FOB) ratio data processing eliminated well-to-well differences in resting baseline signals, reduced error bars, improved curve fits and reduced 95 % confidence interval EC50 and IC50 ranges. FOB ratio data processing decreased variability and improved the precision of repeat measurements in single experimental sessions thereby reducing the minimum threshold difference in EC50 or IC50 values required to distinguish compound potencies. EC50 and IC50 values of TRPM8 agonists and antagonists determined in single experiments were strongly aligned to those from multiple independent experiments. Benchmark TRPM8, TRPV1, and TRPA1 EC50 and IC50 values were within the ranges previously reported for agonist and antagonist standards. The improved precision and accuracy of the TRP Ca2+ mobilization assays afforded by FOB ratio data processing enhances their utility for investigating structure activity relationships. |
| format | Article |
| id | doaj-art-77ed870e947e4bb19afb9229c6df8b6b |
| institution | Kabale University |
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| language | English |
| publishDate | 2025-01-01 |
| publisher | Elsevier |
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| series | SLAS Discovery |
| spelling | doaj-art-77ed870e947e4bb19afb9229c6df8b6b2025-01-23T05:27:26ZengElsevierSLAS Discovery2472-55522025-01-0130100207Optimization and calibration of 384-well kinetic Ca2+ mobilization assays for the human transient receptor potential cation channels TRPM8, TRPV1, and TRPA1David A. Close0V. Blair Journigan1Paul A. Johnston2Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, USADepartment of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, USA; Computational Chemical Genomics Screening Center, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15260, USADepartment of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261, USA; University of Pittsburgh Hillman Cancer Center, Pittsburgh, PA 15232, USA; Corresponding author at: Department of Pharmaceutical Sciences, School of Pharmacy, Salk Hall Room 7402, 3501 Terrace Street, Pittsburgh, PA 15261, USA.Development, optimization, and calibration of human transient receptor potential (TRP) channel Ca2+ mobilization assays for TRPM8, TRPV1, and TRPA1 are described. Heterologous expression of hTRPM8 in HEK293T cells was required for anti-TRPM8 antibody staining and TRPM8 agonist induced Ca2+ mobilization signals which were both used to optimize transfection efficiency. FLIPR Calcium 6 dye concentration, loading time, and TRPM8 transfected cell seeding density were optimized and a DMSO tolerance of ≤0.2 % was set. The resting baseline relative fluorescent unit (RFUs) signals of the TRPM8 Ca2+ mobilization assay exhibited substantial well-to-well variability, even though such differences were small relative to maximal agonist induced responses. Maximum RFU, cumulative RFU sum, or area under the curve values were extracted from Ca2+ mobilization kinetic data to plot curves and calculate EC50 and IC50 values. Fold over baseline (FOB) ratio data processing eliminated well-to-well differences in resting baseline signals, reduced error bars, improved curve fits and reduced 95 % confidence interval EC50 and IC50 ranges. FOB ratio data processing decreased variability and improved the precision of repeat measurements in single experimental sessions thereby reducing the minimum threshold difference in EC50 or IC50 values required to distinguish compound potencies. EC50 and IC50 values of TRPM8 agonists and antagonists determined in single experiments were strongly aligned to those from multiple independent experiments. Benchmark TRPM8, TRPV1, and TRPA1 EC50 and IC50 values were within the ranges previously reported for agonist and antagonist standards. The improved precision and accuracy of the TRP Ca2+ mobilization assays afforded by FOB ratio data processing enhances their utility for investigating structure activity relationships.http://www.sciencedirect.com/science/article/pii/S2472555224000698Human transient receptor potential cation channels TRPM8TRPV1and TRPA1 Kinetic Ca2+ mobilization assaysFold over baseline ratio data processingBenchmarking with known agonists and antagonists |
| spellingShingle | David A. Close V. Blair Journigan Paul A. Johnston Optimization and calibration of 384-well kinetic Ca2+ mobilization assays for the human transient receptor potential cation channels TRPM8, TRPV1, and TRPA1 SLAS Discovery Human transient receptor potential cation channels TRPM8 TRPV1 and TRPA1 Kinetic Ca2+ mobilization assays Fold over baseline ratio data processing Benchmarking with known agonists and antagonists |
| title | Optimization and calibration of 384-well kinetic Ca2+ mobilization assays for the human transient receptor potential cation channels TRPM8, TRPV1, and TRPA1 |
| title_full | Optimization and calibration of 384-well kinetic Ca2+ mobilization assays for the human transient receptor potential cation channels TRPM8, TRPV1, and TRPA1 |
| title_fullStr | Optimization and calibration of 384-well kinetic Ca2+ mobilization assays for the human transient receptor potential cation channels TRPM8, TRPV1, and TRPA1 |
| title_full_unstemmed | Optimization and calibration of 384-well kinetic Ca2+ mobilization assays for the human transient receptor potential cation channels TRPM8, TRPV1, and TRPA1 |
| title_short | Optimization and calibration of 384-well kinetic Ca2+ mobilization assays for the human transient receptor potential cation channels TRPM8, TRPV1, and TRPA1 |
| title_sort | optimization and calibration of 384 well kinetic ca2 mobilization assays for the human transient receptor potential cation channels trpm8 trpv1 and trpa1 |
| topic | Human transient receptor potential cation channels TRPM8 TRPV1 and TRPA1 Kinetic Ca2+ mobilization assays Fold over baseline ratio data processing Benchmarking with known agonists and antagonists |
| url | http://www.sciencedirect.com/science/article/pii/S2472555224000698 |
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