The media of bovine, porcine, and chicken MSCs differentiated for 4 days in the presence of antioxidant supplement (Sigma-Aldrich, A1345) or quercetin was removed and treated with 10 μM 2',7'-dichlorofluorescein (Sigma-Aldrich, St. Louis, MO, USA) for 2 h at 37 °C. After washing twice with PBS, fluorescence was measured (excitation 498 nm and emission 522 nm) using a microplate reader (BioTek, SynergyTM H1 Hybrid Multi-Mode Microplate Reader, Winooski, VT, USA).
Synergy h1 hybrid multi mode microplate reader
Manufactured by Agilent Technologies
Sourced in United States, Italy, United Kingdom, Germany
The Synergy H1 Hybrid Multi-Mode Microplate Reader is a versatile laboratory instrument designed for various microplate-based assays. It combines multiple detection modes, including absorbance, fluorescence, and luminescence, within a single compact unit.
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Lab products found in correlation
Eclipse ti s, by Nikon (5 mentions)
Dual luciferase reporter assay system, by Promega (5 mentions)
Cell proliferation kit 1, by Roche (5 mentions)
96 well plate, by Corning (4 mentions)
Nunc immuno plate, by Thermo Fisher Scientific (3 mentions)
Gen5 data analysis software, by Agilent Technologies (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (3 mentions)
Cell growth determination kit, by Merck Group (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Softmax pro version 7, by Molecular Devices (2 mentions)
Cm h2dcfda dye, by Thermo Fisher Scientific (2 mentions)
Cm h2dcfda, by Agilent Technologies (2 mentions)
Celltiter glo 3d cell viability assay, by Promega (2 mentions)
Quartz microplate, by Hellma (2 mentions)
Boc qar amc, by R&D Systems (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
M5655, by Merck Group (2 mentions)
48 well plate, by Corning (2 mentions)
Salmon sperm dna, by Thermo Fisher Scientific (2 mentions)
276 protocols using synergy h1 hybrid multi mode microplate reader
1
Antioxidant Effects on MSC Oxidative Stress
2
ε-PL Antimicrobial Potency Evaluation
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The ε-PL samples obtained from the fermentation broth of S. albulus WT and the repression strains S1, S2, S3, and S4 were used for the growth inhibition experiments with Bacillus subtilis 168 and E. coli DH5a.
The liquid medium was configured with different ε-PL final concentrations (125 mg/L, 200 mg/L, 250 mg/L, 375 mg/L, and 500 mg/L) based on LB medium. An overnight culture of Bacillus subtilis 168 and E. coli DH5a was added to 96-well plates containing the above medium, and the OD600 of the strain was adjusted to 0.55 to observe the change in its OD600. The OD600 values after 6 h of incubation at 37 °C were used to compare the inhibition potency of ε-PL from different sources to verify the inhibition effect of ε-PL. The OD600 of the medium in 96-well plates was measured in batch using a SynergyTM H1 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA).
The liquid medium was configured with different ε-PL final concentrations (125 mg/L, 200 mg/L, 250 mg/L, 375 mg/L, and 500 mg/L) based on LB medium. An overnight culture of Bacillus subtilis 168 and E. coli DH5a was added to 96-well plates containing the above medium, and the OD600 of the strain was adjusted to 0.55 to observe the change in its OD600. The OD600 values after 6 h of incubation at 37 °C were used to compare the inhibition potency of ε-PL from different sources to verify the inhibition effect of ε-PL. The OD600 of the medium in 96-well plates was measured in batch using a SynergyTM H1 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA).
3
ELISA for Mouse Antibodies
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Sera were obtained from the blood samples by centrifugation at 2775× g at 4 °C for 20 min. We coated 96-well flat-bottom Nunc-Immuno plates (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10 μg/mL of PPD (National Institute for Biological Standards and Control, Hertfordshire, UK) [66 (link)] or goat anti-mouse IgA, IgG1, or IgG2c capture antibody (SouthernBiotech, Birmingham, LA, USA). Sera were diluted at 27 for anti-PPD, and 103 to 107 for IgA, IgG1, and IgG2c, and added to the plates followed by a peroxidase-conjugated anti-mouse total IgG (H+L) (2000-fold dilution, Thermo Fisher Scientific, Inc.) for anti-PPD antibody and peroxidase-conjugated anti-mouse IgG F(ab’)2 (5000-fold dilution, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for IgA, IgG1, and IgG2c. Immunoreactive complexes were detected with 3,3’,5,5’-tetramethylbenzidine (TMB) (BD). The absorbances were measured at 450 nm, using the Synergy H1 Hybrid Multi-Mode Microplate Reader (Agilent Technologies, Inc., Santa Clara, CA, USA). We conducted ELISAs in duplicate wells of 96-well plates.
4
Cell Viability Measurement by Cell Titer Blue
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Cell viability was measured by the Cell Titer Blue assay (Promega, Madison, WI, USA) based on the manufacturer’s guidelines. Briefly, cells were seeded in a 96-wells plate at a density of 5 x 103 cells/well and treated with PMPs (10 PMP/cell) for the indicated time points. Thereafter, 20 μL of Cell Titer Blue substrate was added to each well and incubation under standard cell culture conditions for 1 hour. The plate was read with a Synergy H1 Hybrid Multi-Mode Microplate Reader (Agilent, Santa Clara, CA, USA) at Ex/Em: 560/590 nm via Gen5 data analysis software (Version 2.01).
5
Glycolytic ATP Determination Assay
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Cellular ATP levels were determined by an ATP determination kit (ThermoFisher Scientific) according to manufacturer’s instructions. Briefly, 1 x 104 cells were seeded in 96-well plates with fresh media supplemented with 10% FBS for 1 hour. For glycolytic ATP, cells were treated for 15 min at 37°C with ROT (0.5 μM) and AmA (2.5 μM) to inhibit mitochondrial complexes I and III, respectively. Content of the wells was then collected by centrifugation at 500 x g for 5 min at 4°C, PBS-washed, and centrifuged again. Cells were lysed in 1% Triton for 10 min at room temperature and centrifuged for 5 min at 500 x g. Then, 10 μL of each cell lysate were transferred to another 96-well plate and diluted with 90 μL of the kit’s Luciferine-Luciferase reaction reagent loaded withing the injector of a Synergy H1 Hybrid Multi-Mode Microplate Reader (Agilent). Luminescence was obtained with 1 sec integration times and analyzed with the Gen5 data analysis software (Agilent).
6
Measuring Nutrient Starvation Response
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Once cultures were inoculated into starvation media, OD600 was measured hourly with a Synergy H1 Hybrid Multi-Mode Microplate Reader (Agilent Technologies, Santa Clara, CA, USA) using 300 µL samples in flat-bottom 96-well plates. When necessary, cultures were diluted with similar media to ensure that OD600 readings were in the linear range of the spectrophotometer (0.01 to 0.4). To assess whether single-nutrient starvation conditions had been achieved, after 16 h of incubation in nutrient-starved media, missing nutrients or autoclaved water (control) were added to starved cultures and incubated for an additional 16 h, at which point OD600 measurements were taken.
7
Western Blot Analysis of Protein Extracts
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After cell treatment as above, cells were collected and lysed with RIPA buffer containing protease and phosphatase inhibitors. Alternatively, nuclear and cytoplasmic protein extracts were isolated using the NE-PER nuclear and cytoplasmic extraction reagent kit (Thermo Scientific, Waltham, MA, USA) in accordance with the manufacturer’s instructions. Protein concentration was assessed with the BCA kit (Thermo Scientific, Waltham, MA, USA) and sample absorbance at 750 nm was used to estimate protein concentration using the Synergy H1 Hybrid Multi-Mode Microplate Reader (Biotek, Agilent, Winooski, VT, USA) and data analysis was performed using SoftMax Pro Version 7 (Molecular Devices, Sunnyvale, CA, USA). Western blotting was performed by electrophoresing 20 µg of protein through 8–16% polyacrylamide Mini-PROTEAN pre-cast protein gradient gels (Bio-Rad, Hercules, CA, USA) followed by transfer of proteins to nitrocellulose membranes. Nitrocellulose membranes were incubated overnight at 4 °C with the specific primary antibody (1:1000), and after washing, incubated with goat anti-rabbit IRDye 800 CW and goat anti-mouse IRDye 680 RD (1:5000). Membranes were scanned with LI-COR Odyssey equipment and the intensities of the protein bands were quantified by densitometric analysis using Image Studio 2.0.38 software.
8
Quantifying Mitochondrial ROS in HEK293T Cells
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To analyze levels of ROS production, HEK293T wild-type and HEK293T NDUFA11 knockout cells were cultured for 96 h in DMEM that contained 4.5 g/l glucose supplemented with 10% FBS, 2 mM l -glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 50 μg/ml uridine. HEK293T cells were treated for 68 h with 100 nM rotenone and for the last 24 h before the experiment with NAC or MitoQ as indicated. HEK293T NDUFA11 knockout cells were treated with NAC or MitoQ for 24 h before the ROS measurements where indicated. On the day of the experiment, the cells were incubated on 6 cm dishes for 20 min at 37°C in a CO2 incubator in a culture medium that contained 5 μM red mitochondrial superoxide indicator MitoSOX (Thermo Fisher Scientific; catalogue no. M36008) or 10 μM general oxidative stress indicator CM-H2DCFDA dye (Thermo Fisher Scientific; catalogue no. C6827). Cells were then collected, washed twice, and resuspended in cold PBS at 4°C. Fluorescent signals were corrected for autofluorescence. Fluorescence was measured at an excitation wavelength of 510 nm and emission wavelength of 580 nm for MitoSOX or an excitation wavelength of 495 nm and emission wavelength of 527 nm for CM-H2DCFDA using a fluorescence microplate reader (Synergy H1 Hybrid Multi-Mode Microplate Reader; BioTek).
9
Bacterial Binding Evaluation via Fluorescence
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Suspensions of clinical strains, which are listed in Supplementary Table S1 , were prepared as described in the previous section. The spectrofluorimetric analysis was accomplished according to the protocol described elsewhere (Santos et al., 2020 (link)). Suspensions of bacteria (400 μl) were centrifuged at 9,000 × g for 5 min and resuspended in 40 μl of PB. Then, 20 μl of mCherry-Gp17 at a final concentration of 5 μM was added and incubated at RT for 30 min. Cells were washed twice by centrifugation (9,000 × g for 5 min) using PB to eliminate protein debris and resuspended in 100 μl of PB. The samples were transferred to a black 96-well microplate and examined at a BioTek™ Synergy H1 Hybrid Multi-Mode Microplate Reader with the BioTek Gen5 software. Excitation/emission wavelengths were defined as 570/610 nm (gain 100), and the fluorescence intensity was displayed in arbitrary units (a.u.).
10
PM2.5-induced cytotoxicity in cell lines
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The human bronchial epithelial BEAS-2B cell line and the rat cardio myoblast H9C2 cell line were obtained from the China Infrastructure of Cell Line Resource (Beijing, China) and maintained in DMEM supplemented with 10% FBS and 1% penicillin and streptomycin at 37 °C with 5% CO2. After being cultured for 24 h, the cells were pretreated with PBS or 1 mM metformin for 2 h. Then the culture medium was replaced with serum-free (for BEAS-2B) or 1% FBS (for H9C2) medium and the cells were exposed to freshly dispersed PM2.5 preparations for 24 h. Cell viability was measured with a MTT assay and intracellular ROS levels were determined by a Synergy H1 Hybrid Multi-Mode microplate reader (Biotek Instruments, Inc., Winooski, VT, USA) using DCFH-DA.
To generate a stable AMPKα2-knockdown cell line, 5 × 105 exponentially growing cells were transfected with AMPKα2 shRNA lentivirus for 24 h and then with puromycin (1 μg/mL) for 3 weeks for selection.
To generate a stable AMPKα2-knockdown cell line, 5 × 105 exponentially growing cells were transfected with AMPKα2 shRNA lentivirus for 24 h and then with puromycin (1 μg/mL) for 3 weeks for selection.
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