For quantitative measurement of sebaceous (neutral) lipid content, cells (20 000 cells/well) were cultured in 96‐well “black well/clear bottom” plates (Greiner Bio‐One, Frickenhausen, Germany) in quadruplicates, and were treated with compounds as indicated.24 , 25 , 26 Subsequently, supernatants were discarded, cells were washed twice with phosphate‐buffered saline (PBS; 115 mmol/L NaCl, 20 mmol/L Na2HPO4, pH 7.4; all from Sigma‐Aldrich), and 100 µl of a 1 µg/ml Nile Red (Sigma‐Aldrich) solution in PBS was added to each well. The plates were then incubated at 37°C for 30 minutes, and fluorescence was measured on FlexStation 3 multimode microplate reader (Molecular Devices, San Francisco, CA, USA). Results, measured in relative fluorescence units, are expressed as percentage of the vehicle control regarded as 100%, using 485 nm excitation and 565 nm emission wavelengths.
96 well black well clear bottom plates
Manufactured by Greiner
Sourced in Germany, Austria
The 96-well 'black well/clear bottom' plates are a type of laboratory equipment used for various analytical and experimental purposes. These plates feature black-colored wells with a clear bottom, which allows for optical measurements and detection of fluorescence or luminescence. The core function of these plates is to provide a standardized and versatile platform for cell-based assays, biochemical reactions, and other applications requiring a multi-well format.
Automatically generated - may contain errors
Lab products found in correlation
Flexstation 3, by Molecular Devices (2 mentions)
Cyquant cell proliferation assay kit, by Thermo Fisher Scientific (2 mentions)
Nile red, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Na2hpo4, by Merck Group (1 mentions)
Flexstation 3 multi mode microplate reader, by Molecular Devices (1 mentions)
Mitoprobedilc1 5 assay kit, by Thermo Fisher Scientific (1 mentions)
Envision 2105 multimode plate reader, by PerkinElmer (1 mentions)
Cyquant cytotoxicity assay kit, by Thermo Fisher Scientific (1 mentions)
Prestoblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
Probenecid, by Merck Group (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Flipr, by Molecular Devices (1 mentions)
6 protocols using 96 well black well clear bottom plates
1
Quantification of Sebum Lipid Content
2
Quantifying Cell Proliferation using CyQUANT
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The degree of cellular growth (reflecting proliferation) was determined by measuring the DNA content of cells using CyQUANT Cell Proliferation Assay Kit (Life Technologies Hungary Ltd).24 , 26 , 27 SZ95 sebocytes (2000 cells/well) were cultured in 96‐well “black well/clear bottom” plates (Greiner Bio‐One) and were treated as indicated. Supernatants were then removed by blotting on paper towels, and the plates were subsequently frozen at −80°C. The plates were then thawed at room temperature, and 200 µl of CyQUANT dye/cell lysis buffer mixture was added to each well. After 5 minutes of incubation, fluorescence was measured at 490 nm excitation and 520 nm emission wavelengths using FlexStation 3 multimode microplate reader (Molecular Devices). Relative fluorescence values were expressed as percentage of 24‐hr vehicle control regarded as 100%.
3
Assessing Mitochondrial Membrane Potential
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A decrease in the mitochondrial membrane potential is one of the earliest markers of apoptosis. Therefore, to assess the process, mitochondrial membrane potential of SZ95 sebocytes was determined using a MitoProbe™ DilC1(5) Assay Kit (Life Technologies Hungary Ltd.). 24 , 25 , 26 Cells (20 000 cells/well) were cultured in 96‐well “black well/clear bottom” plates (Greiner Bio One) in quadruplicates and were treated as indicated. After removal of supernatants, cells were incubated for 30 minutes with DilC1(5) working solution (50 μl/well), then washed with PBS, and the fluorescence of DilC1(5) was measured at 630 nm excitation and 670 nm emission wavelengths using FlexStation 3 multi‐mode microplate reader (Molecular Devices). Relative fluorescence values were expressed as percentage of vehicle control regarded as 100%. As a positive control for apoptosis, we applied carbonyl cyanide m‐chlorophenyl hydrazone (CCCP; Life Technologies Hungary Ltd.) dissolved in the DilC1(5) working solution (1:200 for 30 minutes at 37°C).
4
Cytotoxicity Assessment of pCBs
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Cell viability was assessed by CyQUANT™ Cytotoxicity Assay Kit (which measures glucose-6-phosphate dehydrogenase [G6PD] release; λex = 560 nm and λem = 590 nm) and PrestoBlue Cell Viability Reagent (ThermoFisher, λex = 560 nm and λem = 590 nm). Cells were cultured in 96-well black-well/clear-bottom plates (Greiner Bio-One, Kremsmünster, Austria) in quadruplicates and were treated with various concentrations of pCBs over the course of their differentiation. In the case of G6PD release the supernatant was moved to a new plate, while PrestoBlue assay was performed on the remaining cells. In both assays we followed the manufacturer’s instructions. Fluorescence was detected using an EnVision 2105 Multimode Plate Reader (Perkin Elmer, Waltham, MA, USA).
5
Fluorimetric FLIPR Ca2+ Imaging Protocol
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Details of patch clamp protocols and composition of solutions used can be found in the Supplementary Materials and Methods.
Fluorimetric FLIPR Ca 2D imaging Ca 2þ -imaging was performed according to our previously optimized protocol. In brief, keratinocytes were seeded in 96-well black-well/ clear-bottom plates (Greiner Bio-One, Monroe, NC) at a density of 10,000 cells per well. Cells were washed two times with 1% bovine serum albumin (Sigma-Aldrich) and 2.5 mmol/L Probenecid (Sigma-Aldrich) containing Hank's solution (136.8 mmol/L NaCl, 5.4 mmol/ L KCl, 0.34 mmol/L Na 2 HPO 4 , 0.44 mmol/L KH 2 PO 4 , 0.81 mmol/L MgSO 4 , 1.26 mmol/L CaCl 2 , 5.56 mmol/L glucose, 4.17 mmol/L NaHCO 3 ; pH 7.2; Sigma-Aldrich). The cells were then incubated with 1 mmol/L Fluo-4 AM (Life Technologies) containing Hank's solution (100 ml/well) at 37 C for 30 minutes and were then washed three times with Hank's solution (100 ml/well). The plates were then placed in a fluorescent imaging plate reader (Molecular Devices, Sunnyvale, CA), and changes in intracellular calcium concentration (reflected by changes fluorescence; excitation ¼ 490 nm, emission ¼ 520 nm) induced by various concentrations of the compounds were recorded in each well. Experiments were performed in quadruplicate, and the averaged data (and standard error of the mean) were used in the calculations.
Fluorimetric FLIPR Ca 2D imaging Ca 2þ -imaging was performed according to our previously optimized protocol. In brief, keratinocytes were seeded in 96-well black-well/ clear-bottom plates (Greiner Bio-One, Monroe, NC) at a density of 10,000 cells per well. Cells were washed two times with 1% bovine serum albumin (Sigma-Aldrich) and 2.5 mmol/L Probenecid (Sigma-Aldrich) containing Hank's solution (136.8 mmol/L NaCl, 5.4 mmol/ L KCl, 0.34 mmol/L Na 2 HPO 4 , 0.44 mmol/L KH 2 PO 4 , 0.81 mmol/L MgSO 4 , 1.26 mmol/L CaCl 2 , 5.56 mmol/L glucose, 4.17 mmol/L NaHCO 3 ; pH 7.2; Sigma-Aldrich). The cells were then incubated with 1 mmol/L Fluo-4 AM (Life Technologies) containing Hank's solution (100 ml/well) at 37 C for 30 minutes and were then washed three times with Hank's solution (100 ml/well). The plates were then placed in a fluorescent imaging plate reader (Molecular Devices, Sunnyvale, CA), and changes in intracellular calcium concentration (reflected by changes fluorescence; excitation ¼ 490 nm, emission ¼ 520 nm) induced by various concentrations of the compounds were recorded in each well. Experiments were performed in quadruplicate, and the averaged data (and standard error of the mean) were used in the calculations.
6
Quantifying Keratinocyte Proliferation Dynamics
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The degree of cellular growth (reflecting number of viable cells) was determined by measuring the DNA content of cells using CyQUANT Cell Proliferation Assay Kit (Invitrogen, Waltham, MA). Keratinocytes (10,000 cells per well) were cultured in 96-well black-well/clearbottom plates (Greiner Bio-One) in quadruplicate and were treated with various concentrations of TRPV3 agonists. Supernatants were then removed by blotting on paper towels, and the plates were subsequently frozen at e70 C. The plates were then thawed at room temperature, and 200 ml of CyQUANT dye/cell lysis buffer mixture was added to each well. After 5 minutes of incubation, fluorescence was measured at 490 nm excitation and 520 nm emission wavelengths using FLIPR (Molecular Devices).
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