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FLIPRTETRA™ Membrane Potential kit assay development ......G r en lo wb a sck - llcl tt m 1536 p t...
1
Abstract Ion channels are involved in a variety of cellular processes. Dysfunction of ion channels is associated with cardiovascular disease, CNS disorders, and diabetes making them important targets for drug discovery. The gold standard for measuring ion channel activity has been patch-clamp recording. However, this technique requires highly skilled operators, and its very low throughput is a limitation for high-throughput screening (HTS). While automated patch clamping technology such as Molecular Devices IonWorks™HT has set a standard in medium throughput screening, fluorescence-based membrane potential assays provide a excellent option for primary ion channel HTS. The FLIPR TETRA system delivers an entirely new screening platform to address real-time kinetic cell-based assays. Excitation wavelengths have been expanded beyond standard calcium fluorescence, a separate excitation source for membrane potential fluorescence assays. In addition, FLIPR TETRA broadens the FLIPR® system’s capabilities to include simultaneous 536 liquid transfer using Molecular Devices’ novel elastomeric technology. 1536 contact liquid transfer occurs with the same height, speed and volume flexibility offered in traditional FLIPR 96 and 384 pipettors. Here, we developed 384- and 1536-well protocols using the Membrane Potential Blue and Red assay Kits on FLIPR TETRA . Results are compared to assess potential performance differences. Introduction Fluorescence based membrane potential assay is favored for primary screens for its sensitivity, versatility and high throughput capability. FLIPR Membrane Potential Assay Kit (Molecular Devices Corp.) has been shown to outperform the conventional DiBAC 4 (3) assay that uses bis- oxonol fluorescent dye . 1,2 The FLIPR reagent kit offers fast kinetics with an easy mix-and-read assay format; eliminating the cumbersome preparation and temperature sensitivity issues associated with DiBAC. However, as has been previously shown, interaction between a test compound and membrane potential dyes may cause significant change in fluorescence response and lead to altered estimation of compound activity. 3 To minimize potential compound-dye interferences, Molecular Devices offers two Membrane Potential Kits with different formulations. This, in conjunction with FLIPR TETRA technology broadens the range of targets that can be successfully screened using membrane potential as a primary method. FLIPR TETRA System Features FLIPR TETRA utilizes proprietary LED modules with distinct ranges to excite cellular fluorescence. Filtered light is captured using a CCD camera and displayed in real time in the software. LEDs assisted in reducing instrument footprint and facility requirements. In addition, 1536 simultaneous liquid transfer has been added to increase throughput while reducing screening costs. These features are controlled by the drag-and- drop user software which displays data in real-time kinetics. FLIPR TETRA and Membrane Potential Blue and Red Kits: CHO-M1 Membrane Depolarization by KCl in 384 and 1536 Well Format Figure 3. Membrane Depolarization by KCl FLIPR TETRA ™ Membrane Potential kit assay development in 384-well and 1536-well format Carole Crittenden, Jennifer McKie, Jeff Quast, Yen-Wen Chen, and David M. Donofrio Molecular Devices Corporation, Sunnyvale CA Discussion Figure 5. (a.) FLIPR TETRA trace of MP Blue 384-well depolarization of CHO-M1 cell membrane with KCl. (b.) FLIPR TETRA trace of MP Blue 1536-well hyperpolarization of CHO-M1 cell membrane with carbachol Data analysis: Graphs were generated from peak fluorescence values minus the minimum fluorescence. EC 50 values were determined by sigmoidal dose response curve calculation performed in Graph Pad Prism (San Diego, CA). Z’ values were determined at EC 80 concentration 4 . Molecular Devices’ Membrane Potential Red and Blue Assay Kits were used with a membrane potential depolarizing compound and a membrane potential hyperpolarizing compound to compare results between 384-well and 1536-well assays on FLIPR TETRA . Depolarization with KCl gave an increase in signal (Figure 5a). Comparison of graphs between 384-well and 1536-well assays with Red and Blue kits shows values between 14uM and 47uM. Z’ factors at EC 80 were all above 0.5 (Figure 3). Hyperpolarization with carbachol showed a decrease in signal (Figure 5b). Comparison graphs between 384-well and 1536-well assays with Red and Blue kits shows values between 7nM and 200nM. Again Z’ factors at EC 80 were all above 0.71 (Figure 4). In this case, the both the Red and Blue performance was equivalent. Conclusions Availability of two Membrane Potential kits of different formulations broadens applications and increases data quality in HTS primary screening efforts for ion channel targets. Membrane potential assays read on FLIPR TETRA using both kit formulations deliver equivalent results in both 384-well and 1536-well format. References 1. Whiteaker K.L., Gopalakrishnan S.M., Groebe D., Shieh C.-C., Warrior U., Burns D.J., Coghlan M.J., Scott V.E., and Gopalakrishnan M. (2001) Validation of FLIPR Membrane Potential Dye for High Throughput Screening of Potassium Channel Modulators. J. of Biomol. Screen., 6, 305-12. 2. Baxter D.F., Kirk M., Garcia A.F., Raimondi A., Holmqvist M.H., Flint K.K., Bojanic D., Distefano P.S., Curtis R., and Xie Y. (2002) A Novel Membrane Potential-Sensitive Fluorescent Dye Improves Cell-Based Assays for Ion Channels. J. of Biomol. Screen., 7, 79 – 85. 3. Wolff C., Fuks B., and Chatelain P. (2003) Comparative Study of Membrane Potential-Sensitive Fluorescent Probes and Their Use in Ion Channel Screening Assays. J. of Biomol. Screen., 8, 533 – 543. 4. Zhang J. et. al. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays, Journal of Biomolecular Screening, Vol. 4, (2)60-73, (1999) 384 Well Format and Membrane Potential Blue Kit 10 -2 10 -1 10 0 10 1 10 2 10 3 0 200 400 600 800 1000 1200 1400 1600 EC 50 = 48.1uM Z' at EC80 = 0.5 [KCl] uM RLU 384 Well Format and Membrane Potential Blue Kit 10 -4 10 -3 10 -2 10 -1 10 0 10 1 200 400 600 800 Ec 50 = 7.6nM Z' at EC80 = 0.89 [Carbachol] uM Change in RLU 1536 Well Format and Membrane Potential Blue Kit 10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 0 250 500 750 1000 1250 1500 1750 EC 50 = 14.7ul Z' at EC80 = 0.82 [KCl] uM RFU 1536 Well Format and Membrane Potential Blue Kit 10 -4 10 -3 10 -2 10 -1 10 0 0 250 500 750 EC 50 = 8.1nM Z' EC 80 = .71 [Carbachol] uM Change in RFU 1536 Well Format and Membrane Potential Red Kit 10 -2 10 -1 10 0 10 1 10 2 100 200 300 400 500 EC50= 0.2uM Z' EC80 = 0.8 [Carbachol] uM Change in RLU 1536 Well Format and Membrane Potential Red Kit 10 -2 10 -1 10 0 10 1 10 2 10 3 0 1000 2000 3000 4000 5000 EC 50 = 35uM Z' at EC80 = 0..74 [KCl] uM RFU 384 Well Format and Membrane Potential Red Kit 10 -2 10 -1 10 0 10 1 10 2 10 3 0 500 1000 1500 2000 2500 3000 EC 50 = 27.8uM Z' at EC80 = 0.69 [KCl] uM RLU 384 Well Format and Membrane Potential Red Kit 10 -4 10 -3 10 -2 10 -1 10 0 10 1 200 450 700 Ec 50 = 9.7nM Z' at EC80 = 0.88 [Carbachol] uM Change in RLU Materials and Methods Cells used were WT3 CHO-M1 Cell line, Cat# CRL-1985, American Type Culture Collection, Manassas, VA A CHO-K1 cell line transfected with the rat muscarinic M1 acetylcholine receptor. Cells were seeded 1:30 2x per week in Ham’s F12 Media (Cat# 9058, Irvine Scientific, Santa Ana, CA), 10% Fetal Bovine Serum (Cat# SH30071.03, Hyclone, Logan, UT), 1% Pen/Strep/Glutamine (Cat# 10378-016, Invitrogen, Carlsbad, CA), 50ug/ml Geneticin (Cat#10131-035, Invitrogen). Cells were washed with PBS w/o Calcium and Magnesium (Cat#14190-144, Invitrogen) and incubated 5 minutes at 37 o C with Versene 1:5000 (Cat# 9314, Invitrogen). Assay plate preparation: To create 384 well assay plates, cells were re-suspended in culture media and plated at 12,500 cells/well in Costar black- wall clear-bottom 384-well plates (Cat# 3702, Costar, Corning, NY). Plates were incubated overnight at 37 o C in 5%CO 2 . For 1536-well assay plates, 2000 cells /well were plated in Greiner low base black-wall clear-bottom 1536-well plates (Cat# EK-16092, E&K Scientific, Campbell, CA). Plates were incubated overnight at 37 o C in 5%CO 2 . Wash Buffer: Wash buffer was prepared fresh daily. 10X HBSS (Cat#1406- 5056, Invitrogen) was diluted in sterile water for injection (Cat# 9309, Irvine Scientific), and 20mM HEPES (Cat# 15630- 080, Invitrogen) was added. pH was adjusted to 7.4. 384-well FLIPR Membrane Potential red and blue kit protocol: Dye loading buffer for 10 plates was prepared by completely dissolving contents of one bulk-kit reagent vial with 100ml wash buffer. Cell plates were removed from the incubator, then 25ul dye loading buffer added directly to each well without washing. Dye-loaded plates were incubated for 30 minutes at room temperature. 1536-well FLIPR Membrane Potential red and blue kit protocol: Dye loading buffer for 10 plates was prepared by completely dissolving contents of one bulk-kit reagent vial with 67ml wash buffer. Cell plates were removed from the incubator, then 2ul dye loading buffer added directly to each well without washing using an AquaMax ® DW4. Dye-loaded plates were incubated for 30 minutes at room temperature. Membrane potential assay: For 384-well assays, a 5x dose response and for 1536-well assays, a 7x dose response of Carbachol and KCl agonists were prepared in 384 well polypropylene plates. Instrument set up parameters for both plate configurations are shown in Figure 2 below. Plates were read 30 minutes after incubation at room temperature. FLIPR TETRA Setup Parameters for Membrane Potential Figure 2. Instrument Setup parameters Parameter 384 well 1536 well Excitation 510-545 LED Module 510-545 LED Module LED Intensity 50-80% 50-80% m n 5 2 6 - 5 6 5 m n 5 2 6 - 5 6 5 r e t l i F 0 5 - 0 3 0 5 - 0 3 n i a g a r e m a C A / N A / N p o t s F a r e m a C c e S 4 . 0 c e S 4 . 0 e r u s o p x E c e S 1 c e S 1 l a v r e t n I l u 1 l u 5 . 2 1 e m u l o v g n i t t e p i P l u 2 l u 0 4 t h g i e H c e s / l u 5 c e s / l u 5 2 d e e p S Tip up speed 5mm/sec 2mm/sec s e Y s e Y l l e w n i s p i T . b . a Figure 1. FLIPR TETRA data screen FLIPR TETRA and Membrane Potential Blue and Red Kits: CHO-M1 Membrane Hyperpolarization by Carbachol in 384 and 1536 Well Format Figure 4. Membrane Hyperpolarization by Carbachol
Transcript of FLIPRTETRA™ Membrane Potential kit assay development ......G r en lo wb a sck - llcl tt m 1536 p t...
AbstractIon channels are involved in a variety of cellular processes. Dysfunction of ion channels is associated with cardiovascular disease, CNS disorders, and diabetes making them important targets for drug discovery. The gold standard for measuring ion channel activity has been patch-clamp recording. However, this technique requires highly skilled operators, and its very low throughput is a limitation for high-throughput screening (HTS). While automated patch clamping technology such as Molecular Devices IonWorks™HT has set a standard in medium throughput screening, fluorescence-based membrane potential assays provide a excellent option for primary ion channel HTS.
The FLIPRTETRA system delivers an entirely new screening platform to address real-time kinetic cell-based assays. Excitation wavelengths have been expanded beyond standard calcium fluorescence, a separate excitation source for membrane potential fluorescence assays. Inaddition, FLIPRTETRA broadens the FLIPR® system’s capabilities to include simultaneous 536 liquid transfer using Molecular Devices’ novel elastomeric technology. 1536 contact liquid transfer occurs with the same height, speed and volume flexibility offered in traditional FLIPR 96 and 384 pipettors. Here, we developed 384- and 1536-well protocols using the Membrane Potential Blue and Red assay Kits on FLIPRTETRA.Results are compared to assess potential performance differences.
IntroductionFluorescence based membrane potential assay is favored for primary screens for its sensitivity, versatility and high throughput capability. FLIPR Membrane Potential Assay Kit (Molecular Devices Corp.) has been shown to outperform the conventional DiBAC4(3) assay that uses bis-oxonol fluorescent dye .1,2 The FLIPR reagent kit offers fast kinetics with an easy mix-and-read assay format; eliminating the cumbersome preparation and temperature sensitivity issues associated with DiBAC. However, as has been previously shown, interaction between a test compound and membrane potential dyes may cause significant change in fluorescence response and lead to altered estimation of compoundactivity.3 To minimize potential compound-dye interferences, Molecular Devices offers two Membrane Potential Kits with different formulations. This, in conjunction with FLIPRTETRA technology broadens the range of targets that can be successfully screened using membrane potential as a primary method.
FLIPRTETRA System Features
FLIPRTETRA utilizes proprietary LED modules with distinct ranges to excitecellular fluorescence. Filtered light is captured using a CCD camera and displayed in real time in the software. LEDs assisted in reducing instrument footprint and facility requirements. In addition, 1536 simultaneous liquid transfer has been added to increase throughput while reducing screening costs. These features are controlled by the drag-and-drop user software which displays data in real-time kinetics.
FLIPRTETRA and Membrane Potential Blue and Red Kits: CHO-M1 Membrane Depolarization by KCl in
384 and 1536 Well Format
Figure 3. Membrane Depolarization by KCl
FLIPRTETRA™ Membrane Potential kit assay development in384-well and 1536-well format
Carole Crittenden, Jennifer McKie, Jeff Quast, Yen-Wen Chen, and David M. DonofrioMolecular Devices Corporation, Sunnyvale CA
Discussion
Figure 5. (a.) FLIPRTETRA trace of MP Blue 384-well depolarization of CHO-M1 cell membrane with KCl. (b.) FLIPRTETRA trace of MP Blue 1536-well hyperpolarization of CHO-M1 cell membrane with carbachol
Data analysis: Graphs were generated from peak fluorescence values minus the minimum fluorescence. EC50 values were determined by sigmoidal dose response curve calculation performed in Graph Pad Prism (San Diego, CA). Z’ values were determined at EC80 concentration4.
Molecular Devices’ Membrane Potential Red and Blue Assay Kits were used with a membrane potential depolarizing compound and a membrane potential hyperpolarizing compound to compare results between 384-well and 1536-well assays on FLIPRTETRA. Depolarization with KCl gave an increase in signal (Figure 5a). Comparison of graphs between 384-well and 1536-well assays with Red and Blue kits shows values between 14uM and 47uM. Z’ factors at EC80 wereall above 0.5 (Figure 3). Hyperpolarization with carbachol showed a decrease in signal (Figure 5b). Comparison graphs between 384-well and 1536-well assays with Red and Blue kits shows values between 7nM and 200nM. Again Z’ factors at EC80 were all above 0.71 (Figure 4). In this case, the both the Red and Blue performance was equivalent.
Conclusions
Availability of two Membrane Potential kits of different formulations broadens applications and increases data quality in HTS primary screening efforts for ion channel targets. Membrane potential assays read on FLIPRTETRA
using both kit formulations deliver equivalent results in both 384-well and 1536-well format.
References1. Whiteaker K.L., Gopalakrishnan S.M., Groebe D., Shieh C.-C., Warrior U.,
Burns D.J., Coghlan M.J., Scott V.E., and Gopalakrishnan M. (2001) Validation of FLIPR Membrane Potential Dye for High Throughput Screening of Potassium Channel Modulators. J. of Biomol. Screen., 6, 305-12.
2. Baxter D.F., Kirk M., Garcia A.F., Raimondi A., Holmqvist M.H., Flint K.K., Bojanic D., Distefano P.S., Curtis R., and Xie Y. (2002) A Novel Membrane Potential-Sensitive Fluorescent Dye Improves Cell-Based Assays for Ion Channels. J. of Biomol. Screen., 7, 79 – 85.
3. Wolff C., Fuks B., and Chatelain P. (2003) Comparative Study of Membrane Potential-Sensitive Fluorescent Probes and Their Use in Ion Channel Screening Assays. J. of Biomol. Screen., 8, 533 – 543.
4. Zhang J. et. al. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays, Journal of BiomolecularScreening, Vol. 4, (2)60-73, (1999)
384 Well Format and MembranePotential Blue Kit
10-2 10-1 100 101 102 1030
200
400
600
800
1000
1200
1400
1600
EC50= 48.1uMZ' at EC80= 0.5
[KCl] uM
RLU
384 Well Format and MembranePotential Blue Kit
10 -4 10 -3 10 -2 10 -1 10 0 10 1
200
400
600
800
Ec50= 7.6nMZ'at EC80= 0.89
[Carbachol] uM
Cha
nge
in R
LU
1536 Well Format and MembranePotential Blue Kit
10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 30
250
500
750
1000
1250
1500
1750
EC50= 14.7ulZ' at EC80= 0.82
[KCl] uM
RFU
1536 Well Format and MembranePotential Blue Kit
10 -4 10 -3 10 -2 10 -1 10 00
250
500
750
EC50= 8.1nMZ' EC80= .71
[Carbachol] uM
Cha
nge
in R
FU
1536 Well Format and MembranePotential Red Kit
10 -2 10 -1 10 0 10 1 10 2100
200
300
400
500
EC50= 0.2uMZ'EC80= 0.8
[Carbachol] uM
Cha
nge
in R
LU
1536 Well Format and MembranePotential Red Kit
10 -2 10 -1 10 0 10 1 10 2 10 30
1000
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EC50= 35uMZ' at EC80= 0..74
[KCl] uM
RFU
384 Well Format and MembranePotential Red Kit
10 -2 10 -1 10 0 10 1 102 10 30
500
1000
1500
2000
2500
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EC50= 27.8uMZ' at EC80= 0.69
[KCl] uM
RLU
384 Well Format and MembranePotential Red Kit
10 -4 10 -3 10 -2 10 -1 10 0 10 1
200
450
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Ec50= 9.7nMZ'at EC80= 0.88
[Carbachol] uM
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nge
in R
LU
Materials and MethodsCells used were WT3 CHO-M1 Cell line, Cat# CRL-1985, American Type Culture Collection, Manassas, VA A CHO-K1 cell line transfected with the rat muscarinic M1 acetylcholine receptor.
Cells were seeded 1:30 2x per week in Ham’s F12 Media (Cat# 9058, Irvine Scientific, Santa Ana, CA), 10% Fetal Bovine Serum (Cat# SH30071.03, Hyclone, Logan, UT), 1% Pen/Strep/Glutamine (Cat# 10378-016, Invitrogen, Carlsbad, CA), 50ug/ml Geneticin (Cat#10131-035, Invitrogen). Cells were washed with PBS w/o Calcium and Magnesium (Cat#14190-144, Invitrogen) and incubated 5 minutes at 37oCwith Versene 1:5000 (Cat# 9314, Invitrogen).
Assay plate preparation:
To create 384 well assay plates, cells were re-suspended in culture media and plated at 12,500 cells/well in Costar black-wall clear-bottom 384-well plates (Cat# 3702, Costar, Corning, NY). Plates were incubated overnight at 37oC in 5%CO2.
For 1536-well assay plates, 2000 cells /well were plated in Greiner low base black-wall clear-bottom 1536-well plates (Cat# EK-16092, E&K Scientific, Campbell, CA). Plates were incubated overnight at 37oC in 5%CO2.
Wash Buffer:
Wash buffer was prepared fresh daily. 10X HBSS (Cat#1406-5056, Invitrogen) was diluted in sterile water for injection (Cat# 9309, Irvine Scientific), and 20mM HEPES (Cat# 15630-080, Invitrogen) was added. pH was adjusted to 7.4.
384-well FLIPR Membrane Potential red and blue kit protocol:
Dye loading buffer for 10 plates was prepared by completely dissolving contents of one bulk-kit reagent vial with 100ml wash buffer. Cell plates were removed from the incubator, then 25ul dye loading buffer added directly to each well without washing. Dye-loaded plates were incubated for 30 minutes at room temperature.
1536-well FLIPR Membrane Potential red and blue kit protocol:
Dye loading buffer for 10 plates was prepared by completely dissolving contents of one bulk-kit reagent vial with 67ml wash buffer. Cell plates were removed from the incubator, then 2ul dye loading buffer added directly to each well without washing using an AquaMax® DW4. Dye-loaded plates were incubated for 30 minutes at room temperature.
Membrane potential assay:
For 384-well assays, a 5x dose response and for 1536-well assays, a 7x dose response of Carbachol and KCl agonists were prepared in 384 well polypropylene plates. Instrument set up parameters for both plate configurations are shown in Figure 2 below. Plates were read 30 minutes after incubation at room temperature.
FLIPRTETRA Setup Parameters for Membrane Potential
Figure 2. Instrument Setup parameters
Parameter 384 well 1536 wellExcitation 510-545 LED Module 510-545 LED ModuleLED Intensity 50-80% 50-80%
mn526-565mn526-565retliF05-0305-03niag aremaC
A/NA/Npots F aremaCceS 4.0ceS 4.0 erusopxE
ceS 1ceS 1lavretnIlu 1lu 5.21emulov gnittepiPlu2lu 04thgieH
ces/lu5ces/lu52deepSTip up speed 5mm/sec 2mm/sec
seYseYllew ni spiT
.b.a
Figure 1. FLIPRTETRA data screen
FLIPRTETRA and Membrane Potential Blue and Red Kits: CHO-M1 Membrane Hyperpolarization
by Carbachol in 384 and 1536 Well Format
Figure 4. Membrane Hyperpolarization by Carbachol
