Cytokine (IL-6 and IL-10) levels were estimated in both cell supernatants and mouse serum by using ELISA kits (IL-6: #M6000B, IL-10: #M1000B, R&D Systems Inc., Minneapolis, MN, USA) following the manufacturer’s protocols. For IL-6 analyze, cell supernatant was diluted for 1000 times via DMEM-F12 and serum was diluted for 10 times via PBS. For IL-10, both the cell supernatant and mouse serum were not diluted. Briefly, samples were pipetted into a micro-plate pre-coated with capture antibodies and incubated at room temperature for 2 h. After washing 5 times with wash buffer, HRP conjugate antibodies were added and incubated for 2 h at room temperature. Then substrate solution was added and incubated the plates at room temperature protecting from light for 30 min. The reaction was stopped by the stop solution and the optical density of each well was measured at 450 nm with a microplate reader (Multiskan SkyHigh Microplate reader, Thermo Fisher Scientific).
Multiskan skyhigh microplate reader
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The Multiskan SkyHigh microplate reader is a versatile instrument designed for absorbance measurements in microplates. It can perform a wide range of absorbance-based assays, including colorimetric, fluorometric, and luminometric assays.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Peroxidase conjugated secondary antibody, by Vector Laboratories (1 mentions)
Defibrinated sheep blood, by Thermo Fisher Scientific (1 mentions)
Cetylpyridinium chloride, by Merck Group (1 mentions)
Ars solution, by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Nacalai Tesque (1 mentions)
Kg 501, by Selleck Chemicals (1 mentions)
Cck 8 reagent, by Merck Group (1 mentions)
Il 18, by Thermo Fisher Scientific (1 mentions)
Il 18, by Cusabio (1 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
Il 1β, by Cusabio (1 mentions)
Cck 8 solution, by Targetmol (1 mentions)
16 protocols using multiskan skyhigh microplate reader
1
Quantification of Cytokine Levels via ELISA
2
Growth Performance and Organ Weight
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Body weight was recorded for each replicate on d 10 and 14. Average daily gain (ADG) during 1–10 d and 1–14 d was then calculated. The collected liver and spleen were weighed for the measurement of relative organ weight, as calculated by the ratio of organ weight (g) to live body weight (kg) × 100%. Serum diamine oxidase (DAO) activity was measured using a commercial kit (Solarbio Co. Ltd., Beijing, China) with a Multiskan SkyHigh microplate reader (Thermo Fisher Scientific, Waltham, USA).
3
Evaluating Pirfenidone's Impact on Cell Viability
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The cell viability was assessed using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT, Sigma Aldrich, St. Louis, MO, USA) assay reagent after treating with compatible doses of pirfenidone (PFD). Bone marrow macrophages (BMMs) were seeded at a density of 4 × 105/mL in a growth medium (GM) with M-CSF (30 ng/mL) in 96-well plates overnight. The GM was α-MEM (Gibco, Carlsbad, CA, USA) with fetal bovine serum (FBS, 10%, Gibco, Carlsbad, CA, USA) and penicillin/streptomycin (P/S, 1%, Gibco, Carlsbad, CA, USA). Different doses of PFD (0, 100, 200, 400, 800 µM) were then added to the GM, and the samples were incubated for 24 h. The MTT reagent (0.5 mg/mL) was added to each well and cultured at 37 °C for 2 h. The purple formazan crystals indicated complete solubilization by dimethyl sulfoxide (DMSO, Sigma Aldrich, St. Louis, MO, USA), and the absorbance of the samples was measured by using a microplate reader (Multiskan Sky High Microplate Reader, Thermo Fisher, CA, USA) at 562 nm. The cell viability of the mesenchymal stem cells was also analyzed using the MTT assay method and the same protocol with a different cell density (6 × 104/mL) in the GM for 2 and 4 days.
4
Quantifying Matrix Metalloproteinases and Oxidative Stress Markers
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To quantify MMP1, MMP3, MMP9, 8-OHdG, TGF-β1, TGF-β2, and TGF-β3, 96-well microplates were coated with a 100 nM carbonate and bicarbonate-mixed buffer (pH 9.6) and incubated overnight at 4 °C. Microplates were washed with PBS containing 0.1% Tween 20 (TPBS) and blocked with 5% skim milk. Following another PBS wash, each well received 30 μg of protein samples and was incubated overnight at 4 °C. Wells were probed with primary antibodies (Table S3 ) and incubated with peroxidase-conjugated secondary antibodies (Vector Laboratories). For detection, tetramethylbenzidine solution was added for 15–20 min at room temperature, and the reaction was stopped with 2 N sulfuric acid. Optical density was measured at a wavelength of 450 nm with a Multiskan SkyHigh microplate reader (Thermo Fisher Scientific).
5
Erythrocyte Hemolysis Assay with Arachidonic Acid
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Defibrinated sheep blood (ThermoScientific, Oxoid Ltd., Hampshire, UK) was washed several times with sterile PBS (Sartorius) until the supernatant became clear. The blood cells were centrifuged at 1,700 g for 10 min between each wash. Approximately 109 erythrocytes were incubated with 1 ml of various concentrations of AA in PBS supplemented with 1% bovine serum albumin (BSA, VWR Chemicals, Solon, OH, USA) for 1 h at 37°C. The color intensity of the supernatant was determined at 405 nm in a Multiskan SkyHigh microplate reader (ThermoScientific, Life Technologies Holdings Pte Ltd., Singapore). Percentage hemolysis was calculated in comparison to ddw-exploded erythrocytes, which was set to 100% (Sæbø et al., 2023 (link)).
6
BMSCs Mineralization Assay Protocol
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BMSCs were cultured with GM or OM with PFD (0, 100, 200, 400, 800 µM) for 14 days, fixed using 10% formalin for 15 min, and treated with ARS solution (40 mM, pH 4.2, Sigma Aldrich, St. Louis, MO, USA) for 15 min. For quantification, the ARS-stained calcium deposits were extracted for 20 min using sodium phosphate solution (10 mM, pH 7.0), including 10% cetylpyridinium chloride (Sigma Aldrich, St. Louis, MO, USA). Once comprehensive dissolution was attained, the absorbance was measured at a wavelength of 562 nm on a microplate reader (Multiskan SkyHigh Micro-plate Reader, Thermo Fisher, Pleasanton, CA, USA).
7
Cytotoxicity Evaluation of CREB Inhibitors
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MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide) assay kit (Nacalai Tesque, Japan) was used according to the manufacturer’s instructions. 1×103 HEC50B cells in DMEM containing FBS were seeded to each well of 96 well plate. After 24 hours, the medium was replaced to DMEM containing FBS supplemented with CREB inhibitor, KG501 (Selleck, USA) or compoud3i (Selleck, USA), at concentrations of 0.5, 1, 5 and 10 μM. As vehicle control, DMSO was supplemented with DMEM. After 24, 48, 72 and 96 hours, HEC50B cells were incubated in medium with 0.5 mg/mL MTT solution for 2 hours and lysed with 0.04 M HCl in isopropyl alcohol. Absorbance at 570 nm was measured using Multiskan SkyHigh Microplate Reader (Thermo Fisher Scientific).
8
CLTC Gene Knockout Proliferation
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According to the designed sgRNA sequence, the online off-target effect prediction tool (CRISPOR) was used to obtain the top 10 sites that were most likely to be off-target. The 10 sites were amplified by PCR and sequenced. BioEdit software was used to compare the amplified sequence with the wild-type gene sequence to verify the existence of variation.
The wild-type cells and CLTC gene knockout cells were seeded into a 96-well plate with 5 replicates for each kind of cells. Cell proliferation detection reagent MTT Cell Assay Kit (Biotium, USA) was added to the cell supernatant and the absorbance value was detected at 570nm by a Multiskan SkyHigh microplateReader (Thermo Fisher, USA). The OD570 values of the two kinds of cells were compared to determine whether there was a difference in the proliferation activity of the two kinds of cells.
The wild-type cells and CLTC gene knockout cells were seeded into a 96-well plate with 5 replicates for each kind of cells. Cell proliferation detection reagent MTT Cell Assay Kit (Biotium, USA) was added to the cell supernatant and the absorbance value was detected at 570nm by a Multiskan SkyHigh microplateReader (Thermo Fisher, USA). The OD570 values of the two kinds of cells were compared to determine whether there was a difference in the proliferation activity of the two kinds of cells.
9
Cell Viability Assay with CCK-8
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Cells were seeded in 96-well plates, and culture medium was replaced with 100 μL of fresh medium. Ten μL of CCK-8 reagent (Sigma-Aldrich) was added into medium. Cells were incubated for additional 4 hours, and the absorbance (450 nm) was measured with the Multiskan SkyHigh microplate reader (Thermo Fisher Scientific).
10
Quantifying Serum IL-18 and IL-1β
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IL-18 and IL-1β levels in the serum of mice and cell supernatant were evaluated using enzyme-linked immunosorbent assay (ELISA). IL-18 (CUSABIO, Wuhan, China) and IL-1β (CUSABIO) were quantitated according to the manufacturer’s instructions. Multiskan SkyHigh microplate reader (Thermo Fisher Scientific) was used to calculate IL-18 and IL-1β concentrations by forming the standard curve with provided values.
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