mouse neuroblastoma n1e 115 cells Search Results


mouse neuroblastoma derived cell line n1e 115  (ATCC)
95
ATCC mouse neuroblastoma derived cell line n1e 115
Mouse Neuroblastoma Derived Cell Line N1e 115, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 95 stars, based on 1 article reviews
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n1e 115 mouse neuroblastoma cells  (ATCC)
94
ATCC n1e 115 mouse neuroblastoma cells
N1e 115 Mouse Neuroblastoma Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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n1e-115 cells  (JCRB Cell Bank)
90
JCRB Cell Bank n1e-115 cells
N1e 115 Cells, supplied by JCRB Cell Bank, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pei  (Kyfora Bio)
96
Kyfora Bio pei
Pei, supplied by Kyfora Bio, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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ccl  (ATCC)
96
ATCC ccl
Ccl, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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hek293t embryonal kidney cells  (ATCC)
99
ATCC hek293t embryonal kidney cells
A. Localization of the FRET-sensors: <t>N1E-115</t> cells transfected with mTurquoise2Δ-Epac(CD, ΔDEP)- cp173 Venus-Venus (Epac-S H126 ); mTurquoise2Δ-Epac(CD, ΔDEP, Q270E)-td cp173 Venus (Epac-S H187 ); mTurquoise2Δ-Epac(CD, ΔDEP)-td cp173 Dark Venus (Epac-S H159 ) and mTurquoise2Δ-Epac(CD, ΔDEP)- cp174 Cit (Epac-S H147 ). Images were taken 18 hours after transfection. B. FRET efficiency of constructs with different donors. Emission spectra of U2OS cells expressing constructs with donors Cerulaen3 (Epac-S H105 ); mTurquoise (Epac-S H74 ) or mTurquoise2 (Epac-S H126 ) were acquired while exciting at 436 nm. Spectra are normalized to CFP intensity after correction for expression levels (using Venus brightness, excited at 500 nm as detailed in M&M). C. Spectra of constructs with various acceptors. Shown are spectra from N1E-115 cells expressing Epac with acceptor cp173 Venus-Venus (Epac-S H134 ); td cp173 Venus (Epac-S H187 ) or td cp173 Dark Venus (Epac-S H189 ), illuminated with a 442 nm laser. Emission spectra were normalized to isosbestic point at 505 nm. D. Calibration curves for normal and high-affinity sensors. Shown are the average of three independent calibrations performed on cell lysates of <t>HEK293T</t> cells expressing the indicated constructs. cAMP was titrated in under continuous stirring. The sensors were excited at 420 +/- 3 nm and emission was measured at 530 +/- 10 nm and 490 +/- 10 nm for YFP and CFP respectively. Ratios were calculated as YFP over CFP and were normalized between baseline 0% and maximum response 100%. E. In-vivo experiment revealing the difference between high- and normal affinity sensors (Epac-S H126 or Epac-S H134 ) expressed in Hek293T cells. Isoproterenol induces a graded increase in cAMP levels in these cells and was added in increasing amounts as indicated. Signals were normalized between baseline 0% and maximum response 100%. Representative experiment out of 3 repeats. F. Typical FRET time-lapse trace in N1E-115 cells expressing Epac-S H187 . After recording a baseline, at t = 90 s PGE1 (5 μM) was added, and at t = 250 s IBMX (100 μM) and Forskolin (25 μM) were added for calibration. G. A FLIM-FRET time-lapse recording from N1E-115 cells expressing Epac-S H189 . A baseline was followed by addition of IBMX (100 μM) and Forskolin (25 μM) after 140 seconds.
Hek293t Embryonal Kidney Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 99 stars, based on 1 article reviews
hek293t embryonal kidney cells - by Bioz Stars, 2026-09
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poly-l-lysine  (Chemie GmbH)
90
Chemie GmbH poly-l-lysine
A. Localization of the FRET-sensors: <t>N1E-115</t> cells transfected with mTurquoise2Δ-Epac(CD, ΔDEP)- cp173 Venus-Venus (Epac-S H126 ); mTurquoise2Δ-Epac(CD, ΔDEP, Q270E)-td cp173 Venus (Epac-S H187 ); mTurquoise2Δ-Epac(CD, ΔDEP)-td cp173 Dark Venus (Epac-S H159 ) and mTurquoise2Δ-Epac(CD, ΔDEP)- cp174 Cit (Epac-S H147 ). Images were taken 18 hours after transfection. B. FRET efficiency of constructs with different donors. Emission spectra of U2OS cells expressing constructs with donors Cerulaen3 (Epac-S H105 ); mTurquoise (Epac-S H74 ) or mTurquoise2 (Epac-S H126 ) were acquired while exciting at 436 nm. Spectra are normalized to CFP intensity after correction for expression levels (using Venus brightness, excited at 500 nm as detailed in M&M). C. Spectra of constructs with various acceptors. Shown are spectra from N1E-115 cells expressing Epac with acceptor cp173 Venus-Venus (Epac-S H134 ); td cp173 Venus (Epac-S H187 ) or td cp173 Dark Venus (Epac-S H189 ), illuminated with a 442 nm laser. Emission spectra were normalized to isosbestic point at 505 nm. D. Calibration curves for normal and high-affinity sensors. Shown are the average of three independent calibrations performed on cell lysates of <t>HEK293T</t> cells expressing the indicated constructs. cAMP was titrated in under continuous stirring. The sensors were excited at 420 +/- 3 nm and emission was measured at 530 +/- 10 nm and 490 +/- 10 nm for YFP and CFP respectively. Ratios were calculated as YFP over CFP and were normalized between baseline 0% and maximum response 100%. E. In-vivo experiment revealing the difference between high- and normal affinity sensors (Epac-S H126 or Epac-S H134 ) expressed in Hek293T cells. Isoproterenol induces a graded increase in cAMP levels in these cells and was added in increasing amounts as indicated. Signals were normalized between baseline 0% and maximum response 100%. Representative experiment out of 3 repeats. F. Typical FRET time-lapse trace in N1E-115 cells expressing Epac-S H187 . After recording a baseline, at t = 90 s PGE1 (5 μM) was added, and at t = 250 s IBMX (100 μM) and Forskolin (25 μM) were added for calibration. G. A FLIM-FRET time-lapse recording from N1E-115 cells expressing Epac-S H189 . A baseline was followed by addition of IBMX (100 μM) and Forskolin (25 μM) after 140 seconds.
Poly L Lysine, supplied by Chemie GmbH, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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n1e  (DSMZ)
92
DSMZ n1e
A. Localization of the FRET-sensors: <t>N1E-115</t> cells transfected with mTurquoise2Δ-Epac(CD, ΔDEP)- cp173 Venus-Venus (Epac-S H126 ); mTurquoise2Δ-Epac(CD, ΔDEP, Q270E)-td cp173 Venus (Epac-S H187 ); mTurquoise2Δ-Epac(CD, ΔDEP)-td cp173 Dark Venus (Epac-S H159 ) and mTurquoise2Δ-Epac(CD, ΔDEP)- cp174 Cit (Epac-S H147 ). Images were taken 18 hours after transfection. B. FRET efficiency of constructs with different donors. Emission spectra of U2OS cells expressing constructs with donors Cerulaen3 (Epac-S H105 ); mTurquoise (Epac-S H74 ) or mTurquoise2 (Epac-S H126 ) were acquired while exciting at 436 nm. Spectra are normalized to CFP intensity after correction for expression levels (using Venus brightness, excited at 500 nm as detailed in M&M). C. Spectra of constructs with various acceptors. Shown are spectra from N1E-115 cells expressing Epac with acceptor cp173 Venus-Venus (Epac-S H134 ); td cp173 Venus (Epac-S H187 ) or td cp173 Dark Venus (Epac-S H189 ), illuminated with a 442 nm laser. Emission spectra were normalized to isosbestic point at 505 nm. D. Calibration curves for normal and high-affinity sensors. Shown are the average of three independent calibrations performed on cell lysates of <t>HEK293T</t> cells expressing the indicated constructs. cAMP was titrated in under continuous stirring. The sensors were excited at 420 +/- 3 nm and emission was measured at 530 +/- 10 nm and 490 +/- 10 nm for YFP and CFP respectively. Ratios were calculated as YFP over CFP and were normalized between baseline 0% and maximum response 100%. E. In-vivo experiment revealing the difference between high- and normal affinity sensors (Epac-S H126 or Epac-S H134 ) expressed in Hek293T cells. Isoproterenol induces a graded increase in cAMP levels in these cells and was added in increasing amounts as indicated. Signals were normalized between baseline 0% and maximum response 100%. Representative experiment out of 3 repeats. F. Typical FRET time-lapse trace in N1E-115 cells expressing Epac-S H187 . After recording a baseline, at t = 90 s PGE1 (5 μM) was added, and at t = 250 s IBMX (100 μM) and Forskolin (25 μM) were added for calibration. G. A FLIM-FRET time-lapse recording from N1E-115 cells expressing Epac-S H189 . A baseline was followed by addition of IBMX (100 μM) and Forskolin (25 μM) after 140 seconds.
N1e, supplied by DSMZ, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 92 stars, based on 1 article reviews
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plasmid pexj msnorf25 f  (ATCC)
99
ATCC plasmid pexj msnorf25 f
A. Localization of the FRET-sensors: <t>N1E-115</t> cells transfected with mTurquoise2Δ-Epac(CD, ΔDEP)- cp173 Venus-Venus (Epac-S H126 ); mTurquoise2Δ-Epac(CD, ΔDEP, Q270E)-td cp173 Venus (Epac-S H187 ); mTurquoise2Δ-Epac(CD, ΔDEP)-td cp173 Dark Venus (Epac-S H159 ) and mTurquoise2Δ-Epac(CD, ΔDEP)- cp174 Cit (Epac-S H147 ). Images were taken 18 hours after transfection. B. FRET efficiency of constructs with different donors. Emission spectra of U2OS cells expressing constructs with donors Cerulaen3 (Epac-S H105 ); mTurquoise (Epac-S H74 ) or mTurquoise2 (Epac-S H126 ) were acquired while exciting at 436 nm. Spectra are normalized to CFP intensity after correction for expression levels (using Venus brightness, excited at 500 nm as detailed in M&M). C. Spectra of constructs with various acceptors. Shown are spectra from N1E-115 cells expressing Epac with acceptor cp173 Venus-Venus (Epac-S H134 ); td cp173 Venus (Epac-S H187 ) or td cp173 Dark Venus (Epac-S H189 ), illuminated with a 442 nm laser. Emission spectra were normalized to isosbestic point at 505 nm. D. Calibration curves for normal and high-affinity sensors. Shown are the average of three independent calibrations performed on cell lysates of <t>HEK293T</t> cells expressing the indicated constructs. cAMP was titrated in under continuous stirring. The sensors were excited at 420 +/- 3 nm and emission was measured at 530 +/- 10 nm and 490 +/- 10 nm for YFP and CFP respectively. Ratios were calculated as YFP over CFP and were normalized between baseline 0% and maximum response 100%. E. In-vivo experiment revealing the difference between high- and normal affinity sensors (Epac-S H126 or Epac-S H134 ) expressed in Hek293T cells. Isoproterenol induces a graded increase in cAMP levels in these cells and was added in increasing amounts as indicated. Signals were normalized between baseline 0% and maximum response 100%. Representative experiment out of 3 repeats. F. Typical FRET time-lapse trace in N1E-115 cells expressing Epac-S H187 . After recording a baseline, at t = 90 s PGE1 (5 μM) was added, and at t = 250 s IBMX (100 μM) and Forskolin (25 μM) were added for calibration. G. A FLIM-FRET time-lapse recording from N1E-115 cells expressing Epac-S H189 . A baseline was followed by addition of IBMX (100 μM) and Forskolin (25 μM) after 140 seconds.
Plasmid Pexj Msnorf25 F, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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n4tg3 mouse neuroblastoma dsmz acc 101  (DSMZ)
90
DSMZ n4tg3 mouse neuroblastoma dsmz acc 101
A. Localization of the FRET-sensors: <t>N1E-115</t> cells transfected with mTurquoise2Δ-Epac(CD, ΔDEP)- cp173 Venus-Venus (Epac-S H126 ); mTurquoise2Δ-Epac(CD, ΔDEP, Q270E)-td cp173 Venus (Epac-S H187 ); mTurquoise2Δ-Epac(CD, ΔDEP)-td cp173 Dark Venus (Epac-S H159 ) and mTurquoise2Δ-Epac(CD, ΔDEP)- cp174 Cit (Epac-S H147 ). Images were taken 18 hours after transfection. B. FRET efficiency of constructs with different donors. Emission spectra of U2OS cells expressing constructs with donors Cerulaen3 (Epac-S H105 ); mTurquoise (Epac-S H74 ) or mTurquoise2 (Epac-S H126 ) were acquired while exciting at 436 nm. Spectra are normalized to CFP intensity after correction for expression levels (using Venus brightness, excited at 500 nm as detailed in M&M). C. Spectra of constructs with various acceptors. Shown are spectra from N1E-115 cells expressing Epac with acceptor cp173 Venus-Venus (Epac-S H134 ); td cp173 Venus (Epac-S H187 ) or td cp173 Dark Venus (Epac-S H189 ), illuminated with a 442 nm laser. Emission spectra were normalized to isosbestic point at 505 nm. D. Calibration curves for normal and high-affinity sensors. Shown are the average of three independent calibrations performed on cell lysates of <t>HEK293T</t> cells expressing the indicated constructs. cAMP was titrated in under continuous stirring. The sensors were excited at 420 +/- 3 nm and emission was measured at 530 +/- 10 nm and 490 +/- 10 nm for YFP and CFP respectively. Ratios were calculated as YFP over CFP and were normalized between baseline 0% and maximum response 100%. E. In-vivo experiment revealing the difference between high- and normal affinity sensors (Epac-S H126 or Epac-S H134 ) expressed in Hek293T cells. Isoproterenol induces a graded increase in cAMP levels in these cells and was added in increasing amounts as indicated. Signals were normalized between baseline 0% and maximum response 100%. Representative experiment out of 3 repeats. F. Typical FRET time-lapse trace in N1E-115 cells expressing Epac-S H187 . After recording a baseline, at t = 90 s PGE1 (5 μM) was added, and at t = 250 s IBMX (100 μM) and Forskolin (25 μM) were added for calibration. G. A FLIM-FRET time-lapse recording from N1E-115 cells expressing Epac-S H189 . A baseline was followed by addition of IBMX (100 μM) and Forskolin (25 μM) after 140 seconds.
N4tg3 Mouse Neuroblastoma Dsmz Acc 101, supplied by DSMZ, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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n1e-115 cells ecacc #88112303  (DS Pharma Biomedical)
90
DS Pharma Biomedical n1e-115 cells ecacc #88112303
A. Localization of the FRET-sensors: <t>N1E-115</t> cells transfected with mTurquoise2Δ-Epac(CD, ΔDEP)- cp173 Venus-Venus (Epac-S H126 ); mTurquoise2Δ-Epac(CD, ΔDEP, Q270E)-td cp173 Venus (Epac-S H187 ); mTurquoise2Δ-Epac(CD, ΔDEP)-td cp173 Dark Venus (Epac-S H159 ) and mTurquoise2Δ-Epac(CD, ΔDEP)- cp174 Cit (Epac-S H147 ). Images were taken 18 hours after transfection. B. FRET efficiency of constructs with different donors. Emission spectra of U2OS cells expressing constructs with donors Cerulaen3 (Epac-S H105 ); mTurquoise (Epac-S H74 ) or mTurquoise2 (Epac-S H126 ) were acquired while exciting at 436 nm. Spectra are normalized to CFP intensity after correction for expression levels (using Venus brightness, excited at 500 nm as detailed in M&M). C. Spectra of constructs with various acceptors. Shown are spectra from N1E-115 cells expressing Epac with acceptor cp173 Venus-Venus (Epac-S H134 ); td cp173 Venus (Epac-S H187 ) or td cp173 Dark Venus (Epac-S H189 ), illuminated with a 442 nm laser. Emission spectra were normalized to isosbestic point at 505 nm. D. Calibration curves for normal and high-affinity sensors. Shown are the average of three independent calibrations performed on cell lysates of <t>HEK293T</t> cells expressing the indicated constructs. cAMP was titrated in under continuous stirring. The sensors were excited at 420 +/- 3 nm and emission was measured at 530 +/- 10 nm and 490 +/- 10 nm for YFP and CFP respectively. Ratios were calculated as YFP over CFP and were normalized between baseline 0% and maximum response 100%. E. In-vivo experiment revealing the difference between high- and normal affinity sensors (Epac-S H126 or Epac-S H134 ) expressed in Hek293T cells. Isoproterenol induces a graded increase in cAMP levels in these cells and was added in increasing amounts as indicated. Signals were normalized between baseline 0% and maximum response 100%. Representative experiment out of 3 repeats. F. Typical FRET time-lapse trace in N1E-115 cells expressing Epac-S H187 . After recording a baseline, at t = 90 s PGE1 (5 μM) was added, and at t = 250 s IBMX (100 μM) and Forskolin (25 μM) were added for calibration. G. A FLIM-FRET time-lapse recording from N1E-115 cells expressing Epac-S H189 . A baseline was followed by addition of IBMX (100 μM) and Forskolin (25 μM) after 140 seconds.
N1e 115 Cells Ecacc #88112303, supplied by DS Pharma Biomedical, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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c6  (ATCC)
96
ATCC c6
A. Localization of the FRET-sensors: <t>N1E-115</t> cells transfected with mTurquoise2Δ-Epac(CD, ΔDEP)- cp173 Venus-Venus (Epac-S H126 ); mTurquoise2Δ-Epac(CD, ΔDEP, Q270E)-td cp173 Venus (Epac-S H187 ); mTurquoise2Δ-Epac(CD, ΔDEP)-td cp173 Dark Venus (Epac-S H159 ) and mTurquoise2Δ-Epac(CD, ΔDEP)- cp174 Cit (Epac-S H147 ). Images were taken 18 hours after transfection. B. FRET efficiency of constructs with different donors. Emission spectra of U2OS cells expressing constructs with donors Cerulaen3 (Epac-S H105 ); mTurquoise (Epac-S H74 ) or mTurquoise2 (Epac-S H126 ) were acquired while exciting at 436 nm. Spectra are normalized to CFP intensity after correction for expression levels (using Venus brightness, excited at 500 nm as detailed in M&M). C. Spectra of constructs with various acceptors. Shown are spectra from N1E-115 cells expressing Epac with acceptor cp173 Venus-Venus (Epac-S H134 ); td cp173 Venus (Epac-S H187 ) or td cp173 Dark Venus (Epac-S H189 ), illuminated with a 442 nm laser. Emission spectra were normalized to isosbestic point at 505 nm. D. Calibration curves for normal and high-affinity sensors. Shown are the average of three independent calibrations performed on cell lysates of <t>HEK293T</t> cells expressing the indicated constructs. cAMP was titrated in under continuous stirring. The sensors were excited at 420 +/- 3 nm and emission was measured at 530 +/- 10 nm and 490 +/- 10 nm for YFP and CFP respectively. Ratios were calculated as YFP over CFP and were normalized between baseline 0% and maximum response 100%. E. In-vivo experiment revealing the difference between high- and normal affinity sensors (Epac-S H126 or Epac-S H134 ) expressed in Hek293T cells. Isoproterenol induces a graded increase in cAMP levels in these cells and was added in increasing amounts as indicated. Signals were normalized between baseline 0% and maximum response 100%. Representative experiment out of 3 repeats. F. Typical FRET time-lapse trace in N1E-115 cells expressing Epac-S H187 . After recording a baseline, at t = 90 s PGE1 (5 μM) was added, and at t = 250 s IBMX (100 μM) and Forskolin (25 μM) were added for calibration. G. A FLIM-FRET time-lapse recording from N1E-115 cells expressing Epac-S H189 . A baseline was followed by addition of IBMX (100 μM) and Forskolin (25 μM) after 140 seconds.
C6, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


A. Localization of the FRET-sensors: N1E-115 cells transfected with mTurquoise2Δ-Epac(CD, ΔDEP)- cp173 Venus-Venus (Epac-S H126 ); mTurquoise2Δ-Epac(CD, ΔDEP, Q270E)-td cp173 Venus (Epac-S H187 ); mTurquoise2Δ-Epac(CD, ΔDEP)-td cp173 Dark Venus (Epac-S H159 ) and mTurquoise2Δ-Epac(CD, ΔDEP)- cp174 Cit (Epac-S H147 ). Images were taken 18 hours after transfection. B. FRET efficiency of constructs with different donors. Emission spectra of U2OS cells expressing constructs with donors Cerulaen3 (Epac-S H105 ); mTurquoise (Epac-S H74 ) or mTurquoise2 (Epac-S H126 ) were acquired while exciting at 436 nm. Spectra are normalized to CFP intensity after correction for expression levels (using Venus brightness, excited at 500 nm as detailed in M&M). C. Spectra of constructs with various acceptors. Shown are spectra from N1E-115 cells expressing Epac with acceptor cp173 Venus-Venus (Epac-S H134 ); td cp173 Venus (Epac-S H187 ) or td cp173 Dark Venus (Epac-S H189 ), illuminated with a 442 nm laser. Emission spectra were normalized to isosbestic point at 505 nm. D. Calibration curves for normal and high-affinity sensors. Shown are the average of three independent calibrations performed on cell lysates of HEK293T cells expressing the indicated constructs. cAMP was titrated in under continuous stirring. The sensors were excited at 420 +/- 3 nm and emission was measured at 530 +/- 10 nm and 490 +/- 10 nm for YFP and CFP respectively. Ratios were calculated as YFP over CFP and were normalized between baseline 0% and maximum response 100%. E. In-vivo experiment revealing the difference between high- and normal affinity sensors (Epac-S H126 or Epac-S H134 ) expressed in Hek293T cells. Isoproterenol induces a graded increase in cAMP levels in these cells and was added in increasing amounts as indicated. Signals were normalized between baseline 0% and maximum response 100%. Representative experiment out of 3 repeats. F. Typical FRET time-lapse trace in N1E-115 cells expressing Epac-S H187 . After recording a baseline, at t = 90 s PGE1 (5 μM) was added, and at t = 250 s IBMX (100 μM) and Forskolin (25 μM) were added for calibration. G. A FLIM-FRET time-lapse recording from N1E-115 cells expressing Epac-S H189 . A baseline was followed by addition of IBMX (100 μM) and Forskolin (25 μM) after 140 seconds.

Journal: PLoS ONE

Article Title: Fourth-Generation Epac-Based FRET Sensors for cAMP Feature Exceptional Brightness, Photostability and Dynamic Range: Characterization of Dedicated Sensors for FLIM, for Ratiometry and with High Affinity

doi: 10.1371/journal.pone.0122513

Figure Lengend Snippet: A. Localization of the FRET-sensors: N1E-115 cells transfected with mTurquoise2Δ-Epac(CD, ΔDEP)- cp173 Venus-Venus (Epac-S H126 ); mTurquoise2Δ-Epac(CD, ΔDEP, Q270E)-td cp173 Venus (Epac-S H187 ); mTurquoise2Δ-Epac(CD, ΔDEP)-td cp173 Dark Venus (Epac-S H159 ) and mTurquoise2Δ-Epac(CD, ΔDEP)- cp174 Cit (Epac-S H147 ). Images were taken 18 hours after transfection. B. FRET efficiency of constructs with different donors. Emission spectra of U2OS cells expressing constructs with donors Cerulaen3 (Epac-S H105 ); mTurquoise (Epac-S H74 ) or mTurquoise2 (Epac-S H126 ) were acquired while exciting at 436 nm. Spectra are normalized to CFP intensity after correction for expression levels (using Venus brightness, excited at 500 nm as detailed in M&M). C. Spectra of constructs with various acceptors. Shown are spectra from N1E-115 cells expressing Epac with acceptor cp173 Venus-Venus (Epac-S H134 ); td cp173 Venus (Epac-S H187 ) or td cp173 Dark Venus (Epac-S H189 ), illuminated with a 442 nm laser. Emission spectra were normalized to isosbestic point at 505 nm. D. Calibration curves for normal and high-affinity sensors. Shown are the average of three independent calibrations performed on cell lysates of HEK293T cells expressing the indicated constructs. cAMP was titrated in under continuous stirring. The sensors were excited at 420 +/- 3 nm and emission was measured at 530 +/- 10 nm and 490 +/- 10 nm for YFP and CFP respectively. Ratios were calculated as YFP over CFP and were normalized between baseline 0% and maximum response 100%. E. In-vivo experiment revealing the difference between high- and normal affinity sensors (Epac-S H126 or Epac-S H134 ) expressed in Hek293T cells. Isoproterenol induces a graded increase in cAMP levels in these cells and was added in increasing amounts as indicated. Signals were normalized between baseline 0% and maximum response 100%. Representative experiment out of 3 repeats. F. Typical FRET time-lapse trace in N1E-115 cells expressing Epac-S H187 . After recording a baseline, at t = 90 s PGE1 (5 μM) was added, and at t = 250 s IBMX (100 μM) and Forskolin (25 μM) were added for calibration. G. A FLIM-FRET time-lapse recording from N1E-115 cells expressing Epac-S H189 . A baseline was followed by addition of IBMX (100 μM) and Forskolin (25 μM) after 140 seconds.

Article Snippet: Hek293T embryonal kidney cells (American Type Culture Collection crl-1573) N1E-115 mouse neuroblastoma cells (crl-2263) and U2OS human osteosarcoma cells (ATCC HTB-96), a kind gift of Martijn Luijsterburg (LUMC, Leiden, The Netherlands), were cultured in DMEM supplemented with 10% FCS and antibiotics at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Transfection, Construct, Expressing, In Vivo