The viability of cells was estimated using a CCK-8 assay (Cat: A311-01; Vazyme Biotech Co., Ltd., China) according to the instructions provided by the manufacturer. Prior to treatment, the cells were placed in 96-well plates at a density of 5000 cells per well and incubated at 37 °C with 5% CO2 for 24 h. The vehicle solvent dimethyl sulfoxide (DMSO) was added at a concentration of 1 µL/ml or lower, and different concentrations of Ruxo (0 to 180 µM) for a duration of 24 h. Thereafter, CCK8 was introduced, and the cells were incubated at 37 °C and 5% CO2 for 2 h. The absorbance at 450 nm was then measured using Synergy LX Multi-Mode Reader (BioTek Instruments, USA).
Synergy lx multi mode reader
Manufactured by Agilent Technologies
Sourced in United States
The Synergy LX Multi-Mode Reader is a versatile laboratory instrument designed for various detection modes, including absorbance, fluorescence, and luminescence. It is capable of performing a wide range of assays and can be used for a variety of applications in life science research and drug discovery.
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Lab products found in correlation
Celltiter glo 2.0 assay, by Promega (3 mentions)
Hrp conjugated anti human fc antibody, by Jackson ImmunoResearch (2 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (2 mentions)
Celltiter glo luminescent cell viability assay, by Promega (2 mentions)
Realtime glo annexin 5 apoptosis and necrosis kit, by Promega (2 mentions)
Solid white bottom 96 well plates, by Corning (2 mentions)
Cck 8 assay, by Vazyme (1 mentions)
Resazurin dye, by Merck Group (1 mentions)
Lactate assay kit, by Merck Group (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Oxiselect protein carbonyl fluorometric assay kit, by Cell Biolabs (1 mentions)
Griess assay kit, by Promega (1 mentions)
Realtime glo mt cell viability assay, by Promega (1 mentions)
Dynabeads mrna direct purification kit, by Thermo Fisher Scientific (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Rnase free dnase 1, by New England Biolabs (1 mentions)
Dna prep kit, by Illumina (1 mentions)
Phage dna isolation kit, by Norgen Biotek (1 mentions)
Nextseq 2000 platform, by Illumina (1 mentions)
Bcl convert, by Illumina (1 mentions)
50 protocols using synergy lx multi mode reader
1
Evaluating Cell Viability via CCK-8 Assay
2
Cell Viability Assay for ATR Inhibitor
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Cell survival analysis was performed using cell titer-Glo® 2.0 assay (Promega) according to instructions provided by the company. Briefly, tumor cells (2.5 * 103/well) were seeded in 96-well microtiter plates and kept at 37°C in a 5% CO2 incubator. The next day, cells were treated with the indicated concentration of AZD6738 (an ATR inhibitor) or DMSO (control) in the presence or absence of thymidine (6 μM) for 72 or 96 h in the incubator. Next, cells were exposed to cell titer-Glo® 2.0 reagent for 10 min at RT, and the obtained luminescence value was measured by a plate reader (BioTek Synergy/LX multimode reader). The relative cell survival analysis was calculated in relation to DMSO treated samples.
3
Resazurin-Based Cell Viability Assay
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Cell viability was determined as previously described [8 (link)]. For this assay, using the compound resazurin, mitochondrial viability was assessed. Transiently transfected cells were seeded into 96-well plates at a density of approximately 2000 cells per well, and after treatment, resazurin dye (10 µmol/L) was added (Sigma-Aldrich, St. Louis, MO, USA) to each well. Cell viability was tested every 24 h (2, 3, and 4 days after transfection). The plates were incubated for 4 h at 37 °C, and the product was measured at 570 and 600 nm using Biotek Synergy LX Multimode Reader. Each sample was tested in five replicate wells, and three independent experiments were performed.
4
BCA Protein Quantification Assay Protocol
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Total protein content was assessed using the BCA protein assay (Pierce BCA Protein assay cat # 23225, Thermo Fisher Scientific) according to manufacturer’s protocol for the microplate procedure. Various sample dilutions were tested before perform the final quantification test to fall in the linear range of the assay with minimum to no matrix inhibition effect. Samples were loaded neat (20 μL/well) and in triplicates in the 96-well microtiter plate. The standard curve was prepared with BSA in WFI between 25 μg/mL and 2 mg/mL and loaded in duplicate. Upon acquiring the absorbance values at 562 nm wavelength (Synergy LX Multi-Mode Reader, BioTek Instruments Inc.), each standard and sample absorbance value was adjusted for the mean blank value and plotted against the concentration using a best fit curve (Microsoft Excel) to extrapolate the sample values. For the final sample results, five independent assays were performed and averaged. The coefficient of variability across the five assays averaged 8.51% ± 4.17%.
5
SARS-CoV-2 RBD Binding Assay
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ELISA was performed to detect the binding between SARS-CoV-2 RBD and Nanosota-1 drugs (either purified recombinant drugs or drugs in the mouse serum) as previously described (33 ). Briefly, ELISA plates were coated with recombinant SARS-CoV-2 RBD-His or RBD-Fc, and were then incubated sequentially with nanobody drugs, HRP-conjugated anti-llama antibody (1:5,000) (Sigma) or HRP-conjugated anti-human-Fc antibody (1:5,000) (Jackson ImmunoResearch). ELISA substrate (Invitrogen) was added to the plates, and the reactions were stopped with 1N H2SO4. The absorbance at 450 nm (A450) was measured using a Synergy LX Multi-Mode Reader (BioTek).
6
Cell Viability Evaluation Using MTS Assay
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The CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) was used to determine cell viability as described.35 (link),56 (link), 57 (link), 58 (link) Briefly, cells were plated onto 96-well plates with medium containing 10% FBS. After 24 h, the culture medium was replaced with fresh medium containing 5% FBS (control), or the same medium containing different concentrations of metformin. After 48 h, MTS reagent was added into the cell culture. Cells were then incubated at 37°C for an additional 1 h, and the absorbance was measured by a Synergy LX Multi-Mode Reader (Biotek, Winooski, VT, USA).
7
Glucose Consumption and Lactate Production
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Cells (2 × 105/well) were cultured in 6-well plates at 37 °C for 48 h. The glucose content in the medium was detected using a Glucose Assay kit (cat. no. GAGO20; Sigma-Aldrich; Merck KGaA), and the lactate content in the medium was detected using a Lactate Assay kit (cat. no. MAK064; Sigma-Aldrich; Merck KGaA), all according to the manufacturer’s protocols. Samples were analyzed using a Synergy LX Multi-Mode Reader (Biotek, Winooski, VT, USA). The rates of glucose consumption and lactate production were calculated according to the standard curve line and OD value of each sample.
8
Oxidative Stress Assessment in Honey Bees
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The procedures for the lipid and protein assay with the head and thorax tissues, respectively, were the same as described previously (Li-Byarlay et al. 2016 (link), Simone-Finstrom et al. 2016 (link)). In brief, the oxidative damage of lipids was quantified by measuring the malondialdehyde (MDA) level in individual heads. Both the TBARS and the Pierce BCA protein assay kits (Thermo Scientific, Waltham, MA) were used according to the manufacturers’ recommendations. Total soluble protein was determined by the BCA protein assay and used to normalize the corresponding TBARS amounts. Each colony type (feral or managed) included at least 12 individual bees. Thirty-nine foragers from feral colonies and another 39 foragers from managed colonies were tested for the TBARS assay.
For the protein carbonyl assay, an OxiSelect protein carbonyl fluorometric assay kit (no. STA-307, CellBiolabs Inc., San Diego, CA) was used to measure the protein carbonyls in thorax samples. A BioTek Synergy LX multimode reader (BioTek, Winooski, VT) was used for fluorescence.
For the protein carbonyl assay, an OxiSelect protein carbonyl fluorometric assay kit (no. STA-307, CellBiolabs Inc., San Diego, CA) was used to measure the protein carbonyls in thorax samples. A BioTek Synergy LX multimode reader (BioTek, Winooski, VT) was used for fluorescence.
9
Quantifying Nitrate and Nitrite in BALF
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A Griess assay kit (#G2930, Promega, Madison, WI, USA) was used to measure nitrate and nitrite species in BALF samples. All assays were performed at room temperature. A nitrate standard solution (100 µL) was serially diluted (from 100–1.6 µM) in duplicate in a 96-well, flat-bottomed, polystyrene microtiter plate. The diluting medium (PBS) was used as the standard blank. After loading the plate with samples (100 µL), the addition of vanadium (III) chloride (100 µL) to each well was rapidly followed by addition of the freshly mixed Griess reagents, sulfanilamide (50 µL) and N-(1-naphthyl)ethylenediamine dihydrochloride (50 µL). Absorbance was measured at 540 nm using a plate reader Synergy LX multi-mode reader (Biotek, Winooski, VT, USA) following a 30 min incubation period.
10
Cell Viability Assay using RealTime-Glo
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Following treatment, cells were transferred to 96-well plates (MDA-MB-231: 20,000 cells and MCF 10 A: 10,000 cells (as suggested by manufacturer’s protocol)) with fresh-media, as previously20 (link),55 (link). The cells were incubated for 12 h to assess the metabolic activity using RealTime-Glo MT Cell Viability Assay (Promega, USA), as per manufacturer’s protocol. Synergy LX Multi-Mode Reader (BioTek Instruments, USA) was used to record luminescence (Lum) at 1 s integration time. The sample Lum values were normalized with Ctrl to quantify viability using equation (1 ).
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