In our Flp-In-CHO cell line stably expressing the human D2LR, the pERK1/2 response is abrogated by pertussis toxin treatment (data not shown). Thus, this provides a convenient downstream measurement of Gαi/o protein activation. ERK1/2 phosphorylation was measured using the AlphascreenTM SureFire ERK kit (PerkinElmer, Waltham, USA). Cells were seeded into 96-well plates at a density of 50,000 cells/well. After 4–6 h, cells were washed with PBS and incubated in serum-free DMEM (pH 7.4) overnight before assaying. Interaction experiments were performed for each ligand at 37 °C in the presence of 0.1 % ascorbic acid. SB269652 or derivative of interest was pre-equilibrated with cells for 30 min prior to agonist stimulation. Stimulation of the cells was terminated after 5 minutes of agonist exposure by removing the media and the addition of 100 μl of SureFire lysis buffer to each well. The plate was shaken for 5 minutes at room temperature before transferring 5 μl of the lysates to a white 384-well Proxiplate (PerkinElmer, Waltham, USA). Then 8 μl of a 240:1440:7:7 mixture of Surefire activation buffer: Surefire reaction buffer: Alphascreen acceptor beads: Alphascreen donor beads was added to the samples and incubated in the dark at 37 °C for 1.5 h. Plates were read using a Fusion-TM plate reader (PerkinElmer, Waltham, USA).
Fusion plate reader
Manufactured by PerkinElmer
Sourced in United States
The Fusion plate reader is a high-performance multi-mode microplate reader capable of fluorescence, luminescence, and absorbance measurements. It is designed to deliver accurate and reliable results for a wide range of applications in life science research and drug discovery.
Automatically generated - may contain errors
Lab products found in correlation
Proxiplate, by PerkinElmer (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Psicheck 2 vector, by Promega (1 mentions)
Xhoi, by Thermo Fisher Scientific (1 mentions)
Psicheck 2, by GenePharma (1 mentions)
Alphascreen acceptor beads, by PerkinElmer (1 mentions)
Alphascreen surefire p erk assay kit, by PerkinElmer (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Surefire lysis buffer, by PerkinElmer (1 mentions)
Phenol red free dmem, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Tokyo Chemical Industry (1 mentions)
12 protocols using fusion plate reader
1
Measuring Gαi/o Protein Activation via ERK1/2 Phosphorylation
2
ERK1/2 Phosphorylation Assay Protocol
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ERK1/2 phosphorylation was measured using the Alphascreen SureFire ERK kit (PerkinElmer, Waltham, USA). Cells were seeded into 96-well plates at a density of 50,000 cells per well. After 5–7 h, cells were washed with PBS and incubated in serum-free DMEM overnight before assaying. Dose–response experiments were performed for each ligand at 37 °C in the presence of 0.1% ascorbic acid. Stimulation of the cells was terminated after 5 min of agonist stimulation by removing the media and the addition of 100 μl of SureFire lysis buffer to each well. The plate was shaken for 5 min at room temperature (RT) before transferring 5 μl of the lysates to a white 384-well Proxiplate (PerkinElmer, Waltham, USA). Next, 8 μl of a 240:1,440:7:7 mixture of Surefire activation buffer:Surefire reaction buffer:Alphascreen acceptor beads:Alphascreen donor beads was added to the samples and incubated in the dark at 37 °C for 1.5 h. Plates were read using a Fusion-TM plate reader (PerkinElmer, Waltham, USA).
3
Quantification of Chemokine Levels in VSMCs
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Chemokine concentrations of human CCL2 (cat. no. DCP00), CXCL9 (cat. no. DCX900), CXCL10 (cat. no. DIP100) and CXCL11 (cat. no. DCX110) from cultured VSMCs were determined by ELISA assay (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's protocol. Samples and standards were added to the wells and plates were incubated for 2 h at room temperature. Detection solution was added to each well and the plates were further incubated for 2 h at room temperature. Substrate solution was added to each well and plates were incubated for 30 min at room temperature. Stop solution was added to each well and the optical density was detected using a Fusion® plate reader (PerkinElmer, Inc., Waltham, MA, USA) after 30 min.
4
Luciferase Assay for miR-690 Binding
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A 697-bp fragment of the p65 3′UTR containing the predicted binding sites for miR-690 was amplified from mouse genomic DNA using specific primers. Amplicon was cleaved with XhoI and NotI (MBI Fermentas, Burlington, ON, Canada) and cloned in between the XhoI and NotI cleavage sites of psiCHECK-2 vector (Promega, Madison, WI, USA) downstream of the Renilla luciferase reporter gene. The resulting construct was named psiCHECK-2-p65. MiR-690 (50 nM), miR-690-mut (mimics containing a mutated miR-690 seed sequence, 50 nM) (GenePharma), or miR control (50 nM) was co-transfected with psiCHECK-2-p65 reporter (200 ng) into C2C12/Runx2Dox cells using Lipofectamine 2000. After 48 h, firefly and renilla luciferase activity were measured in cell lysates using a Dual-Luciferase Reporter Assay System (Promega) on a Fusion plate reader (Perkin-Elmer life and Analytical Sciences). Firefly luciferase activity was used for normalization and as an internal control for transfection efficiency.
5
Quantifying Cellular cAMP Signaling
6
Kinetic Analysis of ERK1/2 Phosphorylation
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ERK1/2 phosphorylation time course experiments were performed in FlpIn-TREx-HEK293-mGPRC6A cells transfected with G proteins (as above) using the SureFire Alphascreen kit. Cells were plated in 96-well plates; after 6h incubation at 37°C in 5% CO2, cells were washed twice with PBS and incubated in serum- and arginine / lysine-free DMEM at 37°C overnight to reduce basal ERK1/2 phosphorylation. Cells were then stimulated with ligand at various time points and incubated at 37°C in 5% CO2. 10% FBS (v/v) was used as a positive control. The reaction was terminated by removal of compounds and lysis of cells with SureFire lysis buffer (TGR Biosciences). The lysates were then treated as per the SureFire ERK1/2 kit instructions. Plates were incubated in the dark at 37°C for 1 h, then left to equilibrate with the ambient temperature before the fluorescence signal was measured using a Fusion plate reader (PerkinElmer) with standard AlphaScreen settings. Data were normalised to the maximal response elicited by 10% FBS (v/v) at the same time point.
7
Monitoring MAP Kinase Activation by AlphaScreen
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MAP kinase activation was monitored using the bead proximity-based AlphaScreen assay to detect phosphorylated ERK (SureFire p-ERK, PerkinElmer, Boston, MA). Virally transduced HEK-293/EBNA cells were seeded in 96 well plates with serum-free growth medium at 50,000 cells/well, followed by incubation for six hours at 37°C in 7% CO2. Cells were then stimulated with vehicle or agonist for 5 min at 37°C in 7% CO2. Agonist response was terminated following rapid removal of the medium by adding 50 μL per well of SureFire lysis buffer, followed by incubation of cells for 10 min at room temperature. Plates were stored at −80 C prior to analysis for p-ERK levels. Processing of cells for phospho-ERK detection was performed using an AlphaScreen SureFire p-ERK assay kit (PerkinElmer, Boston, MA) according to specifications from the manufacturer. Briefly, 10 μL of lysate were transferred to 384 well plates (OptiPlate) and combined with 17 μL of SureFire buffer containing AlphaScreen beads. Plates were incubated for 2 h at room temperature and the fluorescence signal was recorded using a Fusion plate reader (PerkinElmer), adjusted to standard AlphaScreen settings.
8
miRNA-mediated regulation of Ep300 expression
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2T3 cells were selected for this assay based on their low endogenous expression of miRNAs. 2T3 cells were grown to 85–90% confluence in white 96-well plates in DMEM (Invitrogen) supplemented with 10% FBS, 1% nonessential amino acids, L-glutamine, and penicillin/streptomycin at 37 °C under 5% CO2. Cells were transfected with 20 ng empty PsiCHECK-2-vector, PsiCHECK-2-Ep300 3′ UTR, or PsiCHECK-2-MUT Ep300 3′ UTR for 4 h in reduced serum and antibiotic-free Opti-MEM with Lipofectamine 2000. Cells were co-transfected with the pre-miR-132, inhibitor or a negative control (miR control) (RiboBio) at a concentration of 20 nM, respectively. Firefly and Renilla luciferase were measured in cell lysates using a Dual-Luciferase Reporter Assay System (Promega) on a Fusion plate reader (PerkinElmer, USA). Firefly luciferase activity was used for normalization and as an internal control for transfection efficiency.
9
Quantifying GPCR Activation in CHO Cells
10
Quantitative cAMP Assay Protocol
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