HEK-293T cells were co-transfected with MOR-RLuc (0.25 μg) or Gal1R-RLuc (0.25 μg), as well as with Gαi1-YFP, Gαq-YFP or Gαs-YFP (5 μg) and untagged Gβ1 (4.5 μg) and Gγ2 (5 μg). Experiments were performed approximately 48 hours after transfection. The transient transfected cells were collected, washed, and resuspended in Dulbecco’s PBS (DPBS) with 0.1% glucose and 200 μM sodium bisulfite. Approximately 200,000 cells/well were distributed in 96-well plates, and 5 μM coelenterazine H (NanoLight Technology) was added. Fluorescence of the acceptor was quantified (excitation at 500 nm and emission at 540 nm for 1-s recording) to confirm the constant expression level across experiments. Two minutes after the addition of coelenterazine, increasing concentrations of different MOR or Gal1R agonists were added to different wells. The plate was read after agonist addition using a Mithras LB940 microplate reader (Berthold Technologies). BRET signal from cells was calculated as the ratio of the light emitted by Rluc8 at 485 nm to that emitted by YFP (mVenus variant) at 530 nm. A BRET change was defined as the BRET ratio for the corresponding drug minus the BRET ratio in the absence of the drug. Emax and EC50 values are expressed as the basal subtracted BRET change in the concentration-response graphs.
Mithras lb940 microplate reader
Manufactured by Berthold Technologies
Sourced in Germany
The Mithras LB940 is a microplate reader that can be used to measure the absorbance, luminescence, and fluorescence of samples in microplates. It has a wavelength range of 230 to 850 nm and can accommodate microplates with 6 to 384 wells.
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Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
1 step ultra tmb elisa substrate, by Thermo Fisher Scientific (3 mentions)
Superblock, by Thermo Fisher Scientific (3 mentions)
Toxilight bioassay, by Lonza (3 mentions)
Passive lysis buffer (plb), by Promega (2 mentions)
Half black 96 well plate, by Corning (2 mentions)
Anti dna histone 1 antibody, by Merck Group (2 mentions)
Sytox orange nucleic acid stain, by Thermo Fisher Scientific (2 mentions)
96 well solid bottom white assay microplate, by Corning (2 mentions)
Vialight plus cell proliferation and cytotoxicity bioassay kit, by Lonza (2 mentions)
Coelenterazine h, by Nanolight Technology (1 mentions)
Fluorescein diacetate fda, by Merck Group (1 mentions)
Cytotoxicity detection kitplus, by Roche (1 mentions)
Fcap software, by BD (1 mentions)
Cba human soluble protein master buffer kit, by BD (1 mentions)
Human mmp1 enzyme linked immunosorbent assay elisa kit, by Abcam (1 mentions)
Human il 8 flex set, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Cat elisa kit, by Roche (1 mentions)
Renilla luciferase assay system, by Promega (1 mentions)
40 protocols using mithras lb940 microplate reader
1
BRET Analysis of GPCR Signaling
2
Cytotoxicity Screening of Compounds
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HCT116 and STF293 cells were seeded in 96-well plates (1 × 104/well). After 24 h, fresh medium was added. Five hours later, cells were treated with the different compounds at different concentrations, as indicated. After 20 h, cells were washed twice with PBS and subsequently incubated with 200 µL fluorescein diacetate (FDA) (Sigma, Taufkirchen, Germany) at a concentration of 10 μg/mL in PBS for 30 min at 37 °C. Released fluorescein was quantified with a Mithras LB940 microplate reader (Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany). Cytotoxicity of compounds was analyzed with the Cytotoxicity Detection KitPLUS (Roche, Mannheim, Germany), according to the manufacturer’s instruction, by measuring the release of lactate-dehydrogenase in response to damage of membrane integrity.
3
Quantifying Plasma Anti-CD40 Antibodies
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Detection of recombinant αCD40 in the plasma was assessed by enzyme-linked immunosorbent assay (ELISA). Plates were coated with recombinantly expressed extracellular domain of rhesus CD40 protein fused to maltose binding protein (NHP Reagent Resource) at a concentration of 0.01 mg ml−1 and blocked with Super Block (Thermo Scientific, Woodstock, GA, USA). Pre- and post-treatment samples were serially diluted, plated for 1 h, and washed with PBS/0.05% Tween 20. Circulating αCD40 antibody, 2C10R4, was detected by incubating with polyclonal donkey anti-human IgG (H+L)—horseradish peroxidase (Jackson ImmunoResearch Labs, West Grove, PA, USA). Plates were then incubated with 1-Step Ultra TMB ELISA Substrate (Thermo Scientific). TMB stop solution (Cat# 52-00-01; KPL, Gaithersburg, MD, USA) was added and the absorbance was read on Mithras LB 940 microplate reader (Berthold Technologies, Calmbacher, Germany) at 450 nm. A standard curve was generated with serum spiked with known quantities of αCD40 and was used to calculate the αCD40 concentration present in the serum.
4
Quantification of MMP1 and CXCL8 in MSCs
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MMP1 was quantified by the human MMP1 enzyme-linked immunosorbent assay (ELISA) kit (Abcam, Cambridge, United Kingdom) according to manufacturer’s instructions in undiluted samples. Absorbance was analyzed by a Mithras LB 940 microplate reader (Berthold Technologies). CXCL8 was quantified by cytometric bead array (CBA) using the human IL-8 flex-set and BD CBA human soluble protein master buffer kit (BD Biosciences) following the manufacturer’s protocol. Here, cell culture supernatants were applied in 1:50 dilution to range within the standard curve. Data was acquired on a FACS Canto II flow cytometer and analyzed with FCAP software (BD Biosciences). Protein concentrations produced by 2.5 × 105 MSCs/ml were calculated.
5
Rluc-BmOR3 Internalization Assay
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BRET-based internalization assay was performed as previously described (57 (link)). Briefly, HEK293 cells treated with Ctrl or β-arrestin-1/2 siRNA, or pretransfected with BmArr, were transiently transfected with N-terminal Rluc-fused BmOR3 and plasma membrane marker Lyn-YFP. Twenty-four hours after transfection, the cells were detached and distributed into a 96-well microplate. After another 24-h incubation at 37 °C, the cells were stimulated with vehicle or 1 μM bombykal at 37 °C for 20 min. Rluc substrate coelenterazine h was added at a final concentration of 5 μM before light emissions of YFP and Rluc were recorded using a Mithras LB 940 microplate reader (Berthold Technologies). The BRET signal was calculated as the ratio of emission of YFP to Rluc.
6
Quantification of TGFβ1/Fc Fusion Protein
7
Screening shRNA Effects on Neuronal Activity
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Primary cortical neurons were AAV infected on DIV6 with the pathScreener vector expressing individual hit shRNAs or a non-targeting control (NTC). On DIV10, half of the cultures were silenced with 1 µM TTX, 100 µM D-APV (TTX cocktail) for 48 hrs. On DIV12, the remaining cultures were stimulated for 4 hours with 50 μM BIC, 100 μM 4-AP, 100 μM glycine, 1 μM strychnine (BIC cocktail). Subsequently, all cultures were lysed in passive lysis buffer (Promega). Lysate was transferred into white 96-well assay plates and measurement of luciferase bioluminescence was performed in a Mithras LB 940 Microplate Reader (Berthold Technologies) as described previously26 (link).
8
Evaluating Transcriptional Regulation via CAT Assay
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CES cells seeded in 12-well plates were co-transfected at 70% confluency using Lipofectamine 2000 (Invitrogen) as described by the manufacturer with a mixture of the following plasmids: pM plasmid (20 ng) encoding for proteins in fusion with Gal4 DNA binding domain (GBD-proteins), pG4-TK-CAT reporter plasmid (300 ng) allowing the tethering of the GBD-proteins upstream the TK promoter that controls the expression of the CAT gene, pCi plasmid (200 ng) allowing the expression of additional proteins and pRL-CMV plasmid (20 ng) as control to normalize transfection efficiency. After 24 h culture, the medium was removed and the cells were washed twice with PBS. Cell lysates were prepared and CAT expression was measured using the CAT ELISA kit from Roche according to the manufacturer's instructions. Renilla luciferase luminescence was measured on templates from the same lysates using the Renilla Luciferase Assay System (Promega) according to the manufacturer's protocol and using a Mithras LB940 microplate reader (Berthold technologies).
9
GRN Supplementation Impacts CLL PBMC Viability
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3 × 105 CLL PBMCs in 100 µL were seeded in 96-well luminescence culture plates (white color, Thermo Fisher Scientific, Waltham, MA, USA). In the experimental groups, media were supplemented with 1 or 4 µg/mL recombinant GRN. The CellTiter-Glo luminescent cell viability assays (Promega, Madison, WI, USA) were performed after 48 h according to the manufacturer’s protocol and analyzed using a Mithras LB 940 microplate reader (Berthold Technologies, Bad Wildbad, Germany).
10
Quantification of Phospho-ERK1/2 Levels
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Phosphorylation levels of endogenous extracellular signal-regulated kinases 1/2 (ERK1/2) were detected using the Phospho-ERK1/2 (Thr202/Tyr204) kit (Cisbio Bioassays, France). The transfected cells plated in 96-well plates (Greiner BioOne, UK) were serum-starved for 16 h prior to the experiment in serum-free DMEM media. Afterwards, the cells were stimulated for the indicated times by CB1R ligand diluted in serum-free DMEM and then lysed in 50 μl of supplemented lysis buffer. After homogenization, 16 μl of the cell lysate was transferred from the 96-well plate to a 384-well black plate (Greiner BioOne, UK) and incubated with 4 μl of detection buffer containing anti-ERK1/2-Eu3+cryptate and anti-Phospho-ERK1/2-d2 for at least 4 h in dark. The fluorescence emissions at 665 nm and 620 nm were read on HTRF® compatible Mithras LB 940 microplate reader (Berthold Technologies, Germany). Data are presented as the ratio of 665 nm emission and 620 nm emission multiplied by 10,000.
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