Quantification of melanin in iPSC-derived 3D skin equivalents was performed as described previously [27 ]. Briefly, frozen 3D skin constructs were thawed at room temperature and melanin was extracted using Solvable (PerkinElmer). A 1 mg/ml melanin standard stock solution was prepared by dissolving synthetic melanin (Sigma) in Solvable and a series of melanin standards were prepared from the stock. Solvable was used as the 0.0 mg/ml standard. The tissue samples and the melanin standard series were incubated for at least 16 hours at 60±2°C in a dry bath. The extracted tissue samples and the melanin standards were cooled and centrifuged. Samples or standards were then transferred to the appropriate wells of a 96-well plate (BD Biosciences), and Solvable was added to the wells designated as blanks. The absorbance at 490 nm of each well was measured with a Molecular Devices Vmax plate reader (Molecular Devices). The effect of forskolin on pigmentation in the 3D skin equivalents was determined in triplicate in 3 different experiments. Values are expressed as the mean % increase in melanin above control ± SEM. Statistical analysis was performed using the Student’s t test and significance level has been identified as p<0.05.
Vmax plate reader
Manufactured by Molecular Devices
Sourced in United States
The Vmax plate reader is a versatile instrument designed for absorbance-based assays in a microplate format. It can measure a wide range of absorbance values, providing accurate and reproducible results. The Vmax plate reader is capable of performing various types of absorbance-based assays, such as enzyme-linked immunosorbent assays (ELISAs), cell-based assays, and biochemical assays.
Automatically generated - may contain errors
Lab products found in correlation
Acetylcholinesterase assay kit, by Abcam (4 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (3 mentions)
Pepstatin, by iNtRON Biotechnology (2 mentions)
Leupeptin, by iNtRON Biotechnology (2 mentions)
Aprotinin, by iNtRON Biotechnology (2 mentions)
Free glycerol reagent, by Merck Group (2 mentions)
Synthetic melanin, by Merck Group (1 mentions)
Solvable, by PerkinElmer (1 mentions)
96 well plate, by BD (1 mentions)
Camp elisa kit, by Cell Biolabs (1 mentions)
Tmb substrate, by Merck Group (1 mentions)
Prism 6, by GraphPad (1 mentions)
Isoproterenol hydrochloride, by Merck Group (1 mentions)
Glass homogenizer, by Merck Group (1 mentions)
Acetylcholine assay kit, by Cell Biolabs (1 mentions)
Mouse monoclonal antibody isotyping kit, by Merck Group (1 mentions)
Complement c3 mouse elisa kit, by Abcam (1 mentions)
Diethanolamine, by Thermo Fisher Scientific (1 mentions)
Ache assay kit, by Abcam (1 mentions)
21 protocols using vmax plate reader
1
Melanin Quantification in 3D Skin Equivalents
2
Acetylcholinesterase Activity Assay in Mouse Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The level of AChE activity was determined by using an Acetylcholinesterase Assay Kit (Abcam, Cambridge, UK) in accord with the manufacturer’s protocols. Briefly, the brain cortex from each mouse was homogenized in PRO-PREP protein extraction solution (1.0 mM PMSF, 1.0 mM EDTA, 1.0 μM pepstatin, 1.0 μM leupeptin, and 1.0 μM aprotinin; iNtRON Biotechnology Inc., Seoul, Korea); prior to AChE analysis, the homogenate was stored at −70 °C. For analysis, the homogenate sample (or standards) and the AChE reaction mixture were incubated for 20 min at room temperature in a 96-well plate protected from light. Color alteration within the plate wells was determined by using a Vmax plate reader (Molecular Devices, Sunnyvale, CA, USA) at 405 nm.
3
Acetylcholinesterase Activity Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The AChE activity was determined using an Acetylcholinesterase Assay Kit (Abcam, Cambridge, UK), according to the manufacturer’s protocols. Briefly, the colon of each mouse was homogenized in PRO-PREP protein extraction solution (1.0 mM PMSF, 1.0 mM EDTA, 1.0 µM pepstatin, 1.0 µM leupeptin, and 1.0 µM aprotinin) (iNtRON Biotechnology Inc., Seoul, Korea), after which the homogenates were stored at −70 °C until analysis. The sample or standards and ACh reaction mixture were then incubated in a 96-well plate for 10 min at room temperature, protected from the light. Color alterations were read using a Vmax plate reader (Molecular Devices, Sunnyvale, CA, USA) at 410 nm.
4
Free Glycerol Quantification in 3T3-L1 Adipocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The free glycerol release from the 3T3-L1 adipocytes was determined using a Cell-based Glycerol assay kit (Abcam), as described in a previous study [30 (link)]. Briefly, cell supernatants were collected from 3T3-L1 adipocytes treated with MDI + Vehicle, MDI + OT, and MDI + α-cubebenol, respectively. Subsequently, these samples (25 μL) were mixed with free glycerol reagent (100 μL) and incubated for 15 min at room temperature. Finally, the absorbance of the mixture was observed at 540 nm using a Vmax plate reader (Molecular Devices). The free glycerol concentration was calculated using the following equation:
5
Quantifying Glycerol Release from Adipocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Free glycerol release from primary adipocytes was measured using the free glycerol reagent (Sigma-Aldrich Co.) as described in a previous study [32 (link)]. To measure the glycerol level, primary adipocytes were seeded at a density of 2 × 105 cells/mL in KRBH and cultured in a 37 °C incubator. After 1 h, they were either No (untreated), treated with Vehicle (DMSO), or pretreated with 40 µg/mL of OT or 10, 20, and 30 µg/mL of α-cubebenoate. Following incubation for 24 h, the culture medium was collected from the primary adipocytes, treated with α-cubebenoate and heated at 65 °C for 15 min to inactivate any enzymes released by the adipocytes. The inactivated medium (10 µL) was then mixed with 200 µL of glycerol detection reagent, after which the absorbance was read at 540 nm using a Vmax plate reader (Molecular Devices).
6
cAMP ELISA Assay for Adipocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cAMP concentration was determined using a cAMP ELISA kit (Cell Biolabs INC., San Diego, CA, USA) based on the manufacturer’s instructions. Briefly, the cell lysates were collected from 3T3-L1 adipocytes treated with the MDI + Vehicle, MDI + OT, and MDI + α-cubebenol, respectively. After centrifugation at 3000 rpm for 3 min, each sample was mixed with the labeled AP-conjugate and cAMP complete antibody. The pNpp substrate was added to each well and incubated for 1 h. Finally, the stop solution was added, and the absorbance of each well was read at 540 nm using a Vmax plate reader (Molecular Devices).
7
Evaluating GEGR's Cytoprotective Effects
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HepG2 cells treated as described above were further treated with 5 mM H2O2, then incubated for another 12 h, after which the cell viability was determined using the tetrazolium compound 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Cat. No. M2128, Sigma-Aldrich Co.). To determine cell viability, HepG2 cells were seeded at a density of 5 × 105 cells/2 ml of MEM media and grown for 24 h in a 37°C incubator. When the cells attained 70%–80% confluence, they were either untreated (control group), treated with vehicle (dH2O), or pretreated with 6.25, 12.5, 25, or 50 μg/ml of GEGR dissolved in distilled H2O added to each well. Following incubation for 24 h, the supernatants were discarded, after which 2 ml of fresh MEM media and 500 μl of MTT solution (2 mg/ml in PBS) were added to each well. The cells were then incubated at 37°C for 4 h, after which the formazan precipitate was dissolved in dimethyl sulfoxide (DMSO, Cat. No. D1370.0100, Duchefa Biochemie, Haarlem, the Netherlands), and the absorbance was read at 570 nm directly in the wells using a Vmax plate reader (Molecular Devices).
8
Cytotoxicity Assay with MTS Dye
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At assay termination, the cytotoxicity assay plates are stained with the soluble tetrazolium-based dye MTS (CellTiter Reagent, Promega) to determine cell viability and quantify compound toxicity. MTS is metabolized by the mitochondrial enzymes of metabolically active cells to yield a soluble formazan product, allowing the rapid quantitative analysis of cell viability and compound cytotoxicity. The MTS is a stable solution that does not require preparation before use. At termination of the assay, 15 μL of MTS reagent is added per well. The microtiter plates are then incubated at 37°C for 1.5–2 hrs. The incubation interval was chosen based on empirically determined times for optimal dye reduction. The plates are read spectrophotometrically at 490/650 nm with a Molecular Devices Vmax plate reader.
9
Quantification of C3 Convertase Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The C3 convertase activity was determined using the C3 convertase ELISA Kit (MyBiosource, San Diego, CA, USA), in accordance with the manufacturer’s protocols. Serum from each mouse was subsequently placed in individual wells of a precoated antibody plate, and incubated at 37°C for 90 min, followed by addition of 100 μL biotinylated mouse C3c antibody to each well, and further incubation at 37°C for 60 min. Subsequently, 100 μL of enzyme-conjugate liquid was added to each individual well, and incubated at 37°C for 30 min. Finally, 100 μL of the Color reagent liquid and Color reagent C was added to each well, after which the color alteration was determined by using a Vmax plate reader (Molecular Devices, Sunnyvale, CA, USA) at 450 nm. The concentration of C3c (ng/mL) was determined by standard curve and used to represent C3 convertase activity.
10
Cytokine Quantification in BAL Fluid
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!