The levels of cyclooxygenase (COX)-2, IL-1β, IL-6, tumor necrosis factor (TNF)-α, IL-4, and IL-13 were determined using ELISA Kits (Abcam). The proteins obtained from the mouse muscle samples (50 μg/well) were added to each well of a 96-well plate coated with anti-mouse COX-2, IL-1β, IL-6, TNF-α, IL-4, or IL-13 antibody. After incubation, the wells were washed four times with 300 μL of washing buffer. Subsequently, 100 μL of biotinylated anti-mouse COX-2, IL-1β, IL-6, TNF-α, IL-4, or IL-13 antibody was added to each well, and the plate was incubated for 1 h at room temperature with gentle shaking. The plate was then washed repeatedly, and 100 μL of TMB One-Step Substrate Reagent was added to each well. The plate was subsequently incubated for 30 min at room temperature in the dark with gentle shaking. The reaction was stopped by the addition of 50 μL of stop solution, and the absorbance at 450 nm was immediately measured using a microplate reader (Bio-Rad Model 3550, Bio-Rad, Hercules, CA, USA).
Model 3550
Manufactured by Bio-Rad
Sourced in United States, Japan
The Model 3550 is a microplate reader designed for absorbance measurements. It can be used to perform various assays, including ELISAs, cell-based assays, and other applications that involve the quantification of colored or turbid samples in a microplate format.
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Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Opteia, by BD (3 mentions)
Cell counting kit 8 cck 8, by Beyotime (2 mentions)
Xtt solution, by Sartorius (2 mentions)
96 well plate, by Corning (2 mentions)
Mtt reagent, by Merck Group (2 mentions)
Mtt solution, by Merck Group (2 mentions)
Verteporfin, by Merck Group (1 mentions)
Alp activity assay kit, by Abcam (1 mentions)
Bca protein assay kit, by Abcam (1 mentions)
Ca 2 atpase elisa kit, by MyBioSource (1 mentions)
Mouse il 18 elisa kit, by MBL International Corporation (1 mentions)
Wst 1 cell viability assay kit, by Roche (1 mentions)
Celltiter 96 non radioactive cell proliferation assay, by Promega (1 mentions)
1.5 m 2 amino 2 methyl 1 propanol, by Merck Group (1 mentions)
Model mco 18aic, by Sanyo (1 mentions)
Wst 1, by Roche (1 mentions)
Ifn γ, by BD (1 mentions)
Il 17a, by BioLegend (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
61 protocols using model 3550
1
Quantifying Inflammatory Biomarkers in Muscle
2
Comparative Growth Rates of HGK and HGF
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To compare the growth rates of HGK and HGF cultured in 1% or 18% O 2 , HGK or HGF were seeded at a density of 3.0 × 10 3 per well in 96-well plates (Table 1). Wells with evenly distributed cells were used. After 24 h culture, a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay was performed according to the manufacturer's protocol (ATCC, Manassas, VA, USA). Briefly, HGK or HGF (treated or untreated) were incubated with 10 µL MTT for 2 h and then the MTT reagent was replaced with 100 µL dimethylsulfoxide for another 2 h. Absorbance values for each well were measured by a spectrophotometer at 570 nm (Bio-Rad Model 3550, Bio-Rad, Hercules, CA, USA). For LPS treatment, from a stock solution of 1 mg/mL E. coli LPS in Dulbecco's phosphate-buffered saline, the correct amount of the endotoxin was added to the cell wells to achieve a final concentration of 2 µg/mL at 18% O 2 , 24 h [28] (link).
All experiments were repeated three times at separate occasions, using HGK/HGF explants at the third passage.
All experiments were repeated three times at separate occasions, using HGK/HGF explants at the third passage.
3
SH-SY5Y Cell Viability Assay
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SH-SY5Y cells were treated, and cell viability was determined using the conventional MTS reduction assay. Briefly, after treatment, 40 μl of MTS (2 mg/ml in PBS) was added to each well, and cells were incubated at 37 °C for 4 h. Supernatants were aspirated carefully, and the absorbance at 490 nm was measured (in triplicate) with a microplate reader (BIO-RAD Model 3550, CA, USA). Experiments were repeated three times.
4
Biofilm Inhibition by PAMAM-NH2
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In a physiological state, bacteria tend to exist in biofilms. Bacteria in biofilms are less susceptible to stressful environmental conditions than in their planktonic state. Therefore, minimum biofilm eradication concentration (MBEC) was assessed to evaluate whether PAMAM-NH2 has inhibitory effects on biofilms. MBEC was assessed by microtiter plate assay. The testing was started by growing the biofilm first by incubating the suspension of S. mutans and A. naeslundii for 24 h and E. faecalis for 7 days at 37°C. Then, each microplate well was gently washed three times with PBS to remove unattached bacteria, and PAMAM-NH2 with different concentrations was added. After that, the microplates were incubated for 24 h (S. mutans and A. naeslundii) or 7 days (E. faecalis) at 37°C and were rinsed with PBS. The biofilm was added with 1% of crystal violet solution and cultured at room temperature. The well was gently washed with PBS for three times to remove excess crystal violet and incubated with 95% ethanol by shaking for 15 min. The optical density at 595 nm was measured with a microplate reader (Model 3550, Bio-Rad). Each group was performed in sextuplicate. Three independent batches were conducted for the experiment.
5
Biofilm Inhibition by Chitosan-Polyphenol Nanocomposites
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CPNs were diluted with BHI broth containing 1% sucrose to various concentrations (0-0.9 mM). S. mutans strains (OD 600=1.0) were then cultured in sterile 96-well microtiter plates in BHI broth (containing CPNs and 1% sucrose) at 37°C for 24 h. Sterile water was used as the control group. After incubation, the planktonic bacteria were removed, and the wells were washed three times with PBS. The biofilm was fixed with 100 µL 99.99% methanol per well, and after 15 min, microplates were aspirated and air-dried. Crystal violet (CV) staining was used to measure bacterial biofilm formation 29) . One hundred microliters CV was added to stain the biofilm and incubated at 37°C for 5 min. The excess stain was removed by placing the microplate under running tap water. After air-drying the microplates, dye bound to the adherent cells was resolubilized with 100 µL 33% (v/v) glacial acetic acid per well. The optical density of each well was measured at 570 nm using a microplate reader (Model 3550, Bio-Rad). Biofilm formation by bacteria after co-culturing with CPNs was calculated by Eq. ( 3):
In Eq. ( 3), O control and Omaterial are the optical densities for the control (water) and CPNs groups, respectively.
In Eq. ( 3), O control and Omaterial are the optical densities for the control (water) and CPNs groups, respectively.
6
Measurement of Inflammatory Mediators
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Plasma and cell supernatant were centrifuged and stored at −80°C. IL-6 (cat#D6050), IL-8 (cat#D8000C), TNF-α (cat#DTA00D), IL-1β (cat#DLB50), and MCP-1 (cat#DCP00) levels in supernatant exposed to MSU crystals with or without different inhibitors and the plasma level of TREM-1 (cat#DTRM10C) were analyzed and determined using enzyme-linked immunosorbent assay (ELISA) kits from R&D Systems (USA) in accordance with the manufacturer's instructions. The optical density was measured using a microplate reader (Model 3550, Bio-Rad). A standard curve for IL-1β, IL-6, TNF-α, IL-8, and MCP-1 was established using a known concentration of IL-1β, IL-6, TNF-α, IL-8, and MCP-1 by plotting the optical density relative to the log of the concentration.
7
Toxic Exposure Impact on hiPS-Ch Viability
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KBD-hiPS-Ch and N-hiPS-Ch were seeded in 96-well plates at a density of 1 × 104 cells per well and treated with various concentrations of T-2 toxin (1, 2.5, 5, 10 ng/mL), DON (100, 250, 500, 1000 ng/mL), and 1:100 T-2+DON toxin (1 + 2.5, 2.5 + 250, 5 + 500, 10 + 1000 ng/mL) for 48 h. We used 0.1–10 μM verteporfin (Sigma, Cat.SML0534) to inhibit YAP protein expression in hiPS-Ch for 48 h. Cell viability was then tested using a cell-counting kit (CCK8, NCM Biotech, Suzhou, China). The absorbance at 450 nm was used to calculate relative viability using a microplate reader (Model 3550, Bio-Rad, Hercules, CA, USA).
8
Quantitative ALP Activity Measurement
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To measure ALP activity, we performed in vitro experiments using the protein samples of MC3T3-E1 cell lysates and ALP activity assay kit (Abcam, Cambridge, MA, USA) according to the manufacturer’s manual [19 (link)]. The absorbance of samples in each well of a 96-well plate was determined at an OD of 405 nm on a Bio-Rad Model 3550 microplate reader. Relative ALP activity in experimental µg or µg + CNF1 sample was normalized to the cellular protein content measured using the bicinchoninic acid (BCA) Protein Assay Kit (Abcam, Cambridge, MA, USA) and presented as a percentage of ALP activity in control 1-g sample [50 (link)].
9
Cell Viability and Proliferation Assay
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Cell viability was analyzed using Cell Counting Kit-8 (CCK-8; Beyotime, C0038). Cells were seeded into 96-well plates (7000 cells/well). After treatment with different concentrations of WP1130, the optical density (OD) at 490 nm for each well was obtained using a microplate reader (BIO-RAD Model 3550). Cell proliferation was also analyzed with CCK-8 assay. Cells were seeded into 96-well plates (500 cells/well), then incubated at 37 °C for the next 6 days. From day 1 to day 7, CCK-8 assay was performed conforming to the manufacturer's instructions. The OD value at 450 nm for each well was measured.
10
MTT Assay for Cell Viability
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SH-SY5Y cells were plated at a density of 1 × 104 cells per well in 96-well plates, and the cell viability was determined using the conventional MTT reduction assay. Briefly, after 24 h exposure to H2O2, 4 0 μL of MTT (2 mg/mL in PBS) was added to each well, and the cells were incubated at 37 °C for 4 h. The supernatants were aspirated carefully, and 100 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the precipitate. The absorbance at 570 nm was measured with a microplate reader (BIO-RAD Model 3550, Hercules, CA, USA).
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