Transfection of BHK cells were performed in a 96-well-plate. A total of 90 ng of p16TRE-CMVm-luciferase and 10 ng of phRL-TK (Renilla luciferase expression vector; Promega) were cotransfected into the cells with 50 ng of phTet-On-Flag-NLS-VP16 and 50 ng of phTet-On-Flag-fused cDNAs and incubated for 23 hours in the presence of Dox. Luciferase assays were performed using a Dual-Glo Luciferase Assay system (Promega) in a Wallac ARVO SX 1420 Multilabel Counter (Perkin Elmer Life Sciences) according to the manufacturer’s instructions. The Renilla luciferase activity was used to standardize the transfection efficiency.
Wallac arvo sx 1420 multilabel counter
Manufactured by PerkinElmer
Sourced in United States
The Wallac ARVO SX 1420 Multilabel Counter is a versatile laboratory instrument designed for high-throughput measurement of various sample types. It offers a wide range of detection modes, including luminescence, fluorescence, and absorbance, enabling users to conduct diverse assays and experiments.
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Lab products found in correlation
Dcfh da, by Thermo Fisher Scientific (2 mentions)
Dmem low glucose, by Merck Group (2 mentions)
Dmem high glucose, by Merck Group (2 mentions)
Fluorescein isothiocyanate (fitc), by PerkinElmer (2 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (2 mentions)
Lactic acid, by Merck Group (2 mentions)
Phrl tk, by Promega (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Citrate buffer, by Fujifilm (1 mentions)
Pbs tween, by Fujifilm (1 mentions)
Ht universal chemiluminescent parp assay kit, by Bio-Techne (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
Caspase glo 3 7 reagent, by Promega (1 mentions)
Plus reagent, by Thermo Fisher Scientific (1 mentions)
Luc2p nfat re hygro, by Promega (1 mentions)
Phrl tk vector, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
13 protocols using wallac arvo sx 1420 multilabel counter
1
Inducible Luciferase Assay in BHK Cells
2
Retinal Inflammatory Cytokine Quantification
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The inflammatory cytokines in the retinal tissue were measured using enzyme-linked immunosorbent assay (ELISA) kits for IL-6 and MCP-1 (R&D Systems, Minneapolis, MN, USA). All spectrophotometric readings were performed using an absorption spectrometer (Wallac ARVO SX 1420 Multilabel Counter; PerkinElmer). All procedures were performed according to the manufacturer’s instructions.
3
Human Albumin Sandwich ELISA
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The 100 μl solution containing 1 g/L human albumin primary antibodies (Cat. No. A80-129A; Bethyl, US) were immobilized in each well of 96 well plates by incubation at 37 °C for 3 h. Afterward, the plates were washed with 200 μl/well PBS-Tween (FUJIFILM Wako Pure Chemical, Osaka, Japan), then the 100 μl/well of blocking buffer was added and incubated for 1 h at room temperature. The 100 μl of a serial dilution of the medium sample and standard solution were plated in each well and incubated 2 h at room temperature. The plates were washed with PBS Tween (FUJIFILM Wako Pure Chemical) and 100 μl/well of 1 g/L diluted HRP labeled-human albumin secondary antibodies (Cat. No. A80–129P; Bethyl, US) added and incubated for 2 h at 37OC. To obtain the color reaction, the plates washed by PBS tween and 100 μl of color solution consist of 2.5 mg 0-phenylene diammonium chloride (FUJIFILM Wako Pure Chemical) and 0.5 μl H2O2 (FUJIFILM Wako Pure Chemical) per ml citrate buffer (FUJIFILM Wako Pure Chemical) were added to each well following incubation for 30 min in room temperature. The color reaction was stopped afterward by adding 50 μl/well H2SO4 (4N) (FUJIFILM Wako Pure Chemical) without removing the previous solution. The fluorescence intensity was measured at 490–650 nm fluorescent wavelength by Wallac Arvo SX 1420 multilabel counter (PerkinElmer, Massachusetts, US).
4
PARP-1 Activity Determination in iPSCs
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The activity of PARP-1 was determined following the procedure of the HT Universal Chemiluminescent PARP Assay Kit (Trevigen, Cat. No. 4677-096-K). Briefly, approximately 2 × 106 iPS cells were centrifuged at 400×g for 10 min at 4 °C. The supernatant was discarded and the pellet was suspended in 5–10 pellet volumes of cold 1× PARP buffer containing 0.4 mM PMSF, other protease inhibitors, 0.4 M NaCl, and 1% Triton X-100. The cell suspensions were incubated at 4 °C, with periodic vortexing for 30 min.
The disrupted cell suspensions were centrifuged at 10,000×g for 10 min at 4 °C to remove insoluble material. Forty-microliter aliquots of the supernatant were taken, and 10 μl of the poly (ADP-ribose) glycohydrolase (PARG) inhibitor ADP-HPD (Calbiochem, Cat. No. 118415, 60 μg) was added to each aliquot result for a final concentration of 0.2 M ADP-HPD, and the PARP activity was measured. We had examined appropriate concentrations for PARG inhibition in advance, at 0.05, 0.1, 0.2 and 0.25 M, and determined that 0.2 M would be used for the experiment (data not shown). At least 20 μg of protein per well was used in the assay. All samples in 96-well plates were read using a Wallac ARVO SX 1420 Multilabel Counter (PerkinElmer).
The disrupted cell suspensions were centrifuged at 10,000×g for 10 min at 4 °C to remove insoluble material. Forty-microliter aliquots of the supernatant were taken, and 10 μl of the poly (ADP-ribose) glycohydrolase (PARG) inhibitor ADP-HPD (Calbiochem, Cat. No. 118415, 60 μg) was added to each aliquot result for a final concentration of 0.2 M ADP-HPD, and the PARP activity was measured. We had examined appropriate concentrations for PARG inhibition in advance, at 0.05, 0.1, 0.2 and 0.25 M, and determined that 0.2 M would be used for the experiment (data not shown). At least 20 μg of protein per well was used in the assay. All samples in 96-well plates were read using a Wallac ARVO SX 1420 Multilabel Counter (PerkinElmer).
5
Measuring Reactive Oxygen Species in RPE-Choroid Complex
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ROS were measured in the RPE-choroid complex samples as described previously9 (link)12 (link). Briefly, the eyes were enucleated and the cornea, lens, vitreous and retina were carefully removed; the RPE, together with the choroid, was scraped from the eyecups and placed into 100 μl PBS. The RPE and choroid could not be separated for technical reasons; therefore, RPE-choroid complex samples were used. The samples from both eyes of an individual mouse were mixed and analysed as one sample. These samples were incubated with 1 μl of 0.2 mg/ml cell-permeant fluorescent ROS detection reagent, DCFH-DA (Invitrogen, Carlsbad, CA) at 37 °C, and the fluorescence intensity was measured according to the manufacturer’s protocol, using an absorption spectrometer (Wallac ARVO SX 1420 Multilabel Counter; PerkinElmer, Waltham, MA).
6
Quantifying MCP-1 in Mouse Retina
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Total protein was isolated from mouse retinas. The MCP-1 cytokine content was measured using ELISA kits (R&D Systems, Minneapolis, MN), and a spectrometer (Wallac ARVO SX 1420 Multilabel Counter; Perkin-Elmer). All procedures were performed according to the manufacturers' instructions.
7
Apoptosis Measurement in hiPSC Aggregates
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HiPSC aggregates were formed by inoculating single cells at 2 × 106/well in six-well plates and culturing in a complete medium supplemented with 0.2% free-fatty acid-BSA on a rotary shaker at 120 rpm for 24 h. The formed aggregates were transferred to the dialysis culture system (D/MR(−)) with or without 0.2% FP003 and cultured on a rotary shaker at 120 rpm for 24 h. Afterward, 1:1 ratio of Caspase-Glo® 3/7 reagent (Promega, Wisconsin, USA) was mixed with the aggregates suspension in a 10 ml conical tube (Corning, USA). The mixture was then moved into six-well plates and rotated at 90 rpm at room temperature for 2 h. The serial number of single-cell hiPSCs suspension that was treated with 200 ng/ml Doxorubicin (Sigma-Aldrich) for 24 h was used as a calibrator. The luminescence of each sample was measured by Wallac Arvo SX 1420 multilabel counter (PerkinElmer, Massachusetts, USA).
8
Evaluating FP003 Performance in Dialysis
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The effect of FP003 on device performance was measured by glucose and lactic acid measurements during a 12 h penetration test under cell-free conditions. Low-glucose DMEM (2 mL; Sigma-Aldrich, St. Louis, MO, USA) containing 0.2% FP003 and 0.8 g/L lactic acids (Sigma-Aldrich) was added to the upper culture compartment, and 15 mL of high-glucose DMEM (Sigma-Aldrich) was placed in the lower dialysate compartment. The dialysis-culture system was then placed in a 120-rpm rotary shaker. We collected 50-µm samples of the medium every 2 h and measured changes in glucose and lactic acid concentration using a YSI 2950 multipurpose bioanalyzer (YSI, Yellow Springs, OH, USA).
To test the ability of the device to accumulate macromolecules in the upper culture compartment and differences in permeation of differently sized molecules, 2 µg/mL FITC (Sigma-Aldrich) at different molecular weights (4, 10, and 20 kDa) was added to the upper culture compartment. This experiment was performed using DMEM basal medium in both compartments, similar to the penetration test for macromolecules. Medium samples were collected after 12 h, and FITC concentration was measured using a Wallac Arvo SX 1420 multilabel counter (PerkinElmer, Waltham, MA, USA).
To test the ability of the device to accumulate macromolecules in the upper culture compartment and differences in permeation of differently sized molecules, 2 µg/mL FITC (Sigma-Aldrich) at different molecular weights (4, 10, and 20 kDa) was added to the upper culture compartment. This experiment was performed using DMEM basal medium in both compartments, similar to the penetration test for macromolecules. Medium samples were collected after 12 h, and FITC concentration was measured using a Wallac Arvo SX 1420 multilabel counter (PerkinElmer, Waltham, MA, USA).
9
Measuring cAMP signaling in HEK293 cells
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HEK293 cells were transfected with an empty mammalian expression vector (pME18S) or the full-length BNGR-B2 ORF inserted into the pME18S vector (BNGR-B2/pME18S) using Lipofectamine LTX with PLUS reagent (Invitrogen) according to the manufacturer's instructions. The transfected cells and dissected PGs were treated with a ligand in the presence of 0.5 mM 3-isobutyl-1-methylxanthine. The cAMP assay was performed using the cAMP-Screen Chemiluminescent Immunoassay System (Applied Biosystems) according to the manufacturer's instructions. To extract cAMP from the PG, 100 µl of acidic ethanol (0.1% 10 N HCl, v/v) was added to a gland and vortexed, and then the supernatant was evaporated. The luminescence was measured with a Wallac ARVO SX 1420 Multilabel Counter (PerkinElmer).
10
Comprehensive Hamster Urine Analysis
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The pH of hamster urine was tested using pH test paper BTB (07010060, Advantec, Tokyo, Japan). Glucose, bilirubin, ketone, specific gravity, blood, protein, urobilinogen, nitrite, and leukocyte were measured with N-MULTISTIX® SG-L (Siemens Healthcare Diagnostics Inc., NY). The turbidity of hamster urine was measured using Wallac ARVO sx 1420 multilabel counter (Perkin Elmer, Waltham, MA, USA) at a wavelength of 600 nm.
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