After treated for 24 h, the culture mediums were collected to measure the activity of lactate dehydrogenase (LDH) using the LDH Cytotoxicity Assay Kit (Cayman) according to the manufacturer’s instructions. The absorbance at 490 nm was detected using a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek, USA). Cell growth was measured by the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2-H- tetrazolium bromide) assay. In brief, cells were incubated in 100 μL MTT solution (0.5 mg/ml in RPMI 1640 medium) in 96-well plate for 4-h before the end of incubation. The supernatant was then discarded, and 100 μL DMSO was added to dissolve the colored product (formazan). The absorbance was measured at 540 nm (690 nm as reference) using a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek, USA).
Synergy h4 hybrid multi mode microplate reader
Manufactured by Agilent Technologies
Sourced in United States, Germany
The Synergy H4 Hybrid Multi-Mode Microplate Reader is a versatile laboratory instrument designed for various analytical applications. It combines multiple detection modes, including absorbance, fluorescence, and luminescence, within a single compact unit.
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Lab products found in correlation
Ir700dx nhs ester, by LI COR (4 mentions)
96 well optical black plates, by Thermo Fisher Scientific (4 mentions)
Celltiter glo luminescent cell viability assay kit, by Promega (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ivis lumina xr in vivo imaging system, by PerkinElmer (3 mentions)
Tris base, by Merck Group (2 mentions)
Dneasy blood tissue kit, by Qiagen (2 mentions)
X tremegene hp dna transfection reagent, by Roche (2 mentions)
Enliten atp assay kit, by Promega (2 mentions)
Amplex ultrared, by Thermo Fisher Scientific (2 mentions)
Control sirna, by Horizon Discovery (2 mentions)
Lincode smartpool sirna library human nr lncrna refseq v54, by Horizon Discovery (2 mentions)
Lincode sirna library, by Horizon Discovery (2 mentions)
Dual glo luciferase reporter system, by Promega (2 mentions)
Protein assay kit, by Bio-Rad (2 mentions)
Resazurin, by Merck Group (2 mentions)
Fish dopamine elisa kit, by Cusabio (2 mentions)
Mini vortexer 945300, by Avantor (2 mentions)
Axio observer fluorescent microscopy system, by Zeiss (2 mentions)
158 protocols using synergy h4 hybrid multi mode microplate reader
1
Quantifying Cell Cytotoxicity and Proliferation
2
Luciferase-based Cytotoxicity Assay for CAR T Cells
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Luciferase activity of CBR-labeled CAR T cells (CARM5-CBR and CAR19-CBR; donor ND500) was tested before use in in vitro and in vivo experiments. Therefore, cells were seeded in a 96-well plate, and relative light units were measured using Synergy H4 hybrid multimode microplate reader (BioTek). Unlabeled CAR T cells (CARM5 and CAR19; donor ND517) and R10 medium only were used for control. For luciferase-based killing assay using the luciferase-labeled human leukemia cell line K562-meso/K562-CD19, CAR T cells were added to tumor cells at an effector:target cell ratio of 60:1, 20:1, 6:1, and 2:1, as indicated in the figure legends. Twenty-four hours later, luminescence was measured using Synergy H4 hybrid multimode microplate reader (BioTek). Tumor cells only and medium only were used for controls. Percentage of specific lysis was calculated using the following formula: % specific lysis = 100 × [(experimental data − spontaneous cell death)/(maximum cell death − spontaneous cell death)].
3
Cell Viability Assays for 3D Cultures
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Cell viability assay was performed on cells cultured in 96-well plates, incubated for 3 h in the dark, at 37 °C in a humidified atmosphere with 5% CO 2 , with a buffered solution that contains highly purified resazurin (Sigma-Aldrich). Cell viability was measured as turnover of resazurin to fluorescent resorufin by metabolically active cells. Fluorescence (excitation 530 nm/emission 590 nm) was detected with a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments Inc., VT).
Alternatively, cell viability was determined by quantification of ATP present in metabolically active cells, using the CellTiter-Glo 3D cell viability assay (Promega, WI). The assay was performed according to the manufacturer's instructions in 96-well culture plates (Greiner Bio-One). For quantitative analysis, an ATP standard curve was generated immediately prior to adding the CellTiter-Glo reagent. Luminescence signal was detected using a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments).
Alternatively, cell viability was determined by quantification of ATP present in metabolically active cells, using the CellTiter-Glo 3D cell viability assay (Promega, WI). The assay was performed according to the manufacturer's instructions in 96-well culture plates (Greiner Bio-One). For quantitative analysis, an ATP standard curve was generated immediately prior to adding the CellTiter-Glo reagent. Luminescence signal was detected using a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments).
4
Antiviral Designer Cells Inhibit Viral Spread
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Prevention of virus spread. 5 × 104 immune-like designer cells (ALICECas9/ALICEAb/ALICECas9+Ab) or HEK-293T cells (Control) per well incubated with EGFP-labeled HSV-1 (MOI = 5) for 3 h were then separately seeded on transwell polycarbonate membrane inner chambers with an 8 μm pore size (Corning, cat. no. 3428), and then cocultured with HEK-293T cells (5 × 104 cells/well) cultured for 18 h on transwell outer chambers. After 48 h of co-incubation, the fluorescence intensity of HEK-293T cells on outer chambers was detected by the Synergy™ H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments Inc.).
Prevention of virus infection. HEK-293T cells (5 × 104 cells/well) seeded on the transwell outer chambers, cultured for 18 h, and then infected with EGFP-labeled HSV-1 (MOI = 5) for 3 h. After adding fresh media, the infected cells were cocultured with immune-like designer cells (ALICECas9/ALICEAb/ALICECas9+Ab) or HEK-293T cells (Control) (5 × 104 cells/well) seeded on the transwell polycarbonate membrane inner chambers with an 8-μm pore size (Corning, cat. no. 3428). After 48 h of co-incubation, the fluorescence intensity of individual cells on the transwell polycarbonate membrane inner chambers was detected by the Synergy™ H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments Inc.).
Prevention of virus infection. HEK-293T cells (5 × 104 cells/well) seeded on the transwell outer chambers, cultured for 18 h, and then infected with EGFP-labeled HSV-1 (MOI = 5) for 3 h. After adding fresh media, the infected cells were cocultured with immune-like designer cells (ALICECas9/ALICEAb/ALICECas9+Ab) or HEK-293T cells (Control) (5 × 104 cells/well) seeded on the transwell polycarbonate membrane inner chambers with an 8-μm pore size (Corning, cat. no. 3428). After 48 h of co-incubation, the fluorescence intensity of individual cells on the transwell polycarbonate membrane inner chambers was detected by the Synergy™ H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments Inc.).
5
HepG2 Cell Metabolic Profiling
6
Quantifying Dopamine Levels in Serum and Media
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The dopamine concentrations in serum and culture media were measured using the Dopamine ELISA Kit specific for fish (Cusabio, China, https://www.cusabio.com/ ) according to the manufacturer’s instructions. The 96-well microtiter plate provided in this kit was pre-coated with anti-dopamine monoclonal antibody, and no signification cross-reactivity or interference between dopamine and its analogs was observed. The precision of the assay was determined by the repeated measurement of control or experiment samples. Both positive and negative controls (phosphate buffer solution (PBS)-only and blank, respectively) were analyzed in this assay. After all reactions, the optical density of each well was measured at 450 nm within 10 min of reaction termination using a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek, USA). The dopamine concentration of each sample was determined according to a standard curve.
7
Extracellular ATP Release after TPt Treatment
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The extracellular
release of ATP postTPt treatment was determined with
an ATP chemiluminescence assay kit (Beyotime, Shanghai, China). 4T1
cells were plated at 15 000 cells/well overnight and then treated
with SZU101, oxaliplatin orTPt at a dose of 100 μM
for 24 h. After incubation, the cell media were collected and centrifuged
at 2500 rpm × 10 min at 4 °C. The level of ATP in the supernatants
was measured by the commercial kit according to manufacturer’s
protocol. The final chemiluminescence was recorded on a Synergy H4
Hybrid Multi-Mode Microplate Reader (Bio-Tek).
release of ATP post
an ATP chemiluminescence assay kit (Beyotime, Shanghai, China). 4T1
cells were plated at 15 000 cells/well overnight and then treated
with SZU101, oxaliplatin or
for 24 h. After incubation, the cell media were collected and centrifuged
at 2500 rpm × 10 min at 4 °C. The level of ATP in the supernatants
was measured by the commercial kit according to manufacturer’s
protocol. The final chemiluminescence was recorded on a Synergy H4
Hybrid Multi-Mode Microplate Reader (Bio-Tek).
8
Seminal Antioxidant Capacity Evaluation
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Total antioxidant capacity (TAC) was measured by
colorimetry using a total antioxidant assay kit (MBL,
Germany). TAC was analyzed by means of a Microplate
Reader (Synergy™ H4 Hybrid Multi-Mode Microplate
Reader, BioTek®, USA) and calculated as nmol/μl of
semen. Briefly, frozen seminal plasma was thawed by
placing vials into a water bath at 37˚C for 20 minutes,
and immediately assessed for its antioxidant capacity
following the manufacturer’s instructions (17 (link)).
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Total antioxidant capacity (TAC) was measured by
colorimetry using a total antioxidant assay kit (MBL,
Germany). TAC was analyzed by means of a Microplate
Reader (Synergy™ H4 Hybrid Multi-Mode Microplate
Reader, BioTek®, USA) and calculated as nmol/μl of
semen. Briefly, frozen seminal plasma was thawed by
placing vials into a water bath at 37˚C for 20 minutes,
and immediately assessed for its antioxidant capacity
following the manufacturer’s instructions (17 (link)).
9
Biodistribution of Antioxidant Enzymes
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Male Kunming mice were randomly divided into 3 groups (n = 5 mice per group). The first group (CON) was untreated. The second group (SOD) was injected with 0.5 mL of FITC-labeled SOD (2 kU/mL) intraperitoneally and the other group (GST-TAT-SOD) received intraperitoneal injection of 0.5 mL of the FITC-labeled recombinant protein GST-TAT-SOD (2 kU/mL). Three hours after injection, all animals were sacrificed by cervical dislocation. The liver, spleen, lung, brain, and kidney were weighed and 10% homogenates were prepared with ice-cold saline using a homogenizer, respectively. The fluorescence of tissue homogenates from organs was determined with a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT). To normalize fluorescence, the protein contents of the 10% homogenates were determined by BCA Protein Assay Kit (Thermo, USA) following manufacturer's directions. Fluorescence was corrected for background signal and normalized for protein content and expressed as fluorescence/μg of protein.
10
Quantifying C9-Flu Binding Kinetics
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In C9-Flu binding experiments, 15 nM of C9-Flu was titrated with increasing concentrations of protein. In competitive titration experiments, unlabeled C9 was used to compete with C9-Flu for binding to RecQ constructs. Fluorescence anisotropy was measured in a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek).
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