A total of 4.0×103 cells per well were seeded in 96-well plates, and cell viability was assessed using the Cell Counting kit-F (CK06, Dojindo, Japan) at 24 h, 48 h, and 72 h after incubation under normoxia (20% O2) or hypoxia (1% O2). The hypoxia-resistant rate was assessed by the ratio of the proliferation rate under hypoxia to that under normoxia at each time point. The fluorescence intensity was measured by SH-9000lab (Corona Electric, Ibaraki, Japan) on a plate reader at an excitation wavelength of 490 nm and emission wavelength of 515 nm.
Sh 9000lab
Manufactured by Corona Electric
Sourced in Japan
The SH-9000Lab is a high-performance laboratory equipment that serves as a reliable solution for various scientific and research applications. It is designed to deliver consistent and accurate results, catering to the needs of researchers and laboratory professionals.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Mem medium, by Thermo Fisher Scientific (2 mentions)
Wst 8 solution, by Dojindo Laboratories (2 mentions)
Enzyme linked immunosorbent assay kit, by R&D Systems (2 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (2 mentions)
Jmp pro, by SAS Institute (2 mentions)
Cell counting kit f, by Dojindo Laboratories (1 mentions)
Bz 8100, by Keyence (1 mentions)
Violamo, by As One (1 mentions)
Cs 5100, by Sysmex (1 mentions)
Wst 8, by Dojindo Laboratories (1 mentions)
Svcam 1, by R&D Systems (1 mentions)
Pai 1, by R&D Systems (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Xn 9000, by Sysmex (1 mentions)
Mmp 9, by R&D Systems (1 mentions)
Aba elisa kit, by Cusabio (1 mentions)
Dpph antioxidant assay kit, by Dojindo Laboratories (1 mentions)
Sod assay kit wst, by Dojindo Laboratories (1 mentions)
Gallic acid, by Fujifilm (1 mentions)
32 protocols using sh 9000lab
1
Hypoxia-Resistant Cell Proliferation Assay
2
Stratum Corneum Evaluation Protocol
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The stratum corneum water content was measured, and the stratum corneum was collected at baseline and after 8 weeks by the tape stripping method. The trypsin activity in the stratum corneum was measured. Furthermore, the degree of desmoglein 1 localization in the stratum corneum was also scored.
a) Stratum corneum water content measurement The stratum corneum water content was measured following the same procedure as in Experiment 1.
b) Analysis of the stratum corneum 13 14
Trypsin activity
Desmoglein 1
3
Cytotoxicity Screening of Natural Compounds
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Human fibroblast cells, MRC-5, were plated on 96-well flat bottom plates at a density of 1.5 × 104 cells/well with 100 μl of MEM medium (Life Technologies, Grand Islands, NY, USA) containing 10% fetal bovine serum (Hana-nesco Bio, Tokyo, Japan) and 1% penicillin-streptomycin (Life Technologies) and incubated at 37°C with 5% CO2 for 2 days. Test compounds in 5 μl of 50% DMSO–H2O and 100 μl of MEM medium were mixed and added to each well. After 2 days cultivation at 37°C with 5% CO2, cell density and morphological changes were observed under a microscope. After observation, 10 μl of WST-8 solution (Dojindo, Kumamoto, Japan) was added to the cells and the plate was incubated at 37°C with 5% CO2 for 2 h. Then, absorbance at 450 nm was measured by spectrophotometer (SH-9000Lab, Corona Electric). Cytotoxicity was measured in duplicate. Staurosporine (our product, in Kitasato Natural Products Library) was used as a positive (cytotoxic) compound. The concentration range of test compounds are: 1.6 to 50 μg/ml (final concentration) for kerriamycin B, kerriamycin C, and aggreticin; 0.075 to 10 μg/ml for deacetylkinamycin C, deoxyfrenolicin, nanaomycin A, and naphthacemycin A9; 3.1 to 200 μg/ml for tetracycline and patulin; 9.4 to 75 μg/ml for exophillic acid. IC50 values were calculated by the equation described in the reference (Arita-Morioka et al., 2015 (link)).
4
Cell Viability Assay of Fibroblasts
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Human fibroblast cells, MRC-5, were plated on 96-well flat bottom plates at a density of 1.5 × 10 4 cells/well with 100 µl of MEM medium (Life Technologies, Grand Islands, NY, USA) containing 10% fetal bovine serum (Hana-nesco Bio, Tokyo, Japan) and 1% penicillin-streptomycin (Life Technologies) and incubated at 37°C with 5% CO 2 for 48 h. Approximately 100 µl of MEM medium, containing 5 µl of each compound dissolved in 50% (v/v) aqueous DMSO, was added to each well. After 48 h cultivation at 37°C with 5% CO 2 , cell density and morphological changes were observed under a microscope. After observation, 10 µl of WST-8 solution (Dojindo, Kumamoto, Japan) was added to the cells and the plate was incubated at 37°C with 5% CO 2 for 2 h. The absorbance was measured at 450 nm by spectrophotometer (SH-9000 Lab., Corona Electric, Ibaraki, Japan). The growth of MRC-5 cells was measured in three independent experiments.
5
Horse Chestnut Seed Antioxidant Capacity
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A test liquid was prepared by extracting 25 mg of a ground part of horse chestnut seed with 1 mL of distilled water in a 1.5-mL microtube (Violamo, AS ONE Corporation, Osaka, Japan) at 100 °C for 10 min. A fluorescence plate reader (SH-9000Lab, CORONA ELECTRIC Co., Ltd., Ibaragi, Japan) kept at 37 °C was used to measure fluorescence intensity of fluorescein after we placed 35 lL of a test liquid or a Trolox solution and then 115 lL of a fluorescein solution (94.4 nM/75 mM-phosphate buffer solution, pH 7.4) in a well of a 96-well microplate (#3072, Becton-Dickinson). Then, 8 min after addition of 50 lL of an 2,2 0 -azobis-2-methyl-propanimidamide, dihydrochloride (AAPH) solution (31.7 mM/75 mM-phosphate buffer solution, pH 7.4) and shaking, the microplate reader was used to take measurements at intervals of 2 min for 90 min to register the time course of fluorescence intensity. An excitation wavelength of 485 nm and a detection wavelength of 520 nm were used for measurement on the microplate reader. Hydroxyl Radical Antioxidant Capacity (H-ORAC) values were expressed in Trolox equivalents (mmol TE/g).
6
Measuring Worm Autofluorescence as Aging Marker
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We recently developed a method to measure autofluorescence as an aging marker for individual worms (28 (link)). In brief, 6-day-old worms fed OP50 or FC from 0 days were washed with M9 buffer and placed into 1.0 μL of M9 buffer on a 384-well black plate (Stem, Tokyo, Japan) covered with Saran Wrap (Asahi Kasei, Tokyo, Japan). The blank (M9 buffer only) data were checked three times for each well, considering the fluctuation across wells. Minimal detection limits and quantifiable limits were determined on the basis of blank data on each day as μ (mean of the blank) + 3.29σ (standard deviation) and μ + √2 × 10σ, respectively. The autofluorescence in the worm body was captured using a multimode grating microplate reader model SH-9000Lab (excitation, 340 nm; emission, 430 nm; Corona Electric, Ibaraki, Japan). After measurement, each worm was individually maintained on a 4-cm-diameter plate covered with OP50 or FC (2 mg/10 μL M9 buffer) at 25°C. The data on worms that died in 2 days were excluded because the autofluorescence could be due to death and not from AGEs (62 (link)). The assay was performed with more than 20 worms. Three biological replicates were analyzed for this study.
7
Platelet, Cytokine, and Coagulation Markers
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Total platelet counts were determined with an automated cell counter (XN9000). The serum concentrations of IL-6 and plasma concentrations of sTF were measured with an enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, Minn). Plasma concentrations of fibrin/fibrinogen degradation products (FDPs) and d -dimer were measured with latex immunoassay (CS5100; Sysmex Corporation). Frozen samples were thawed and subsequently processed according to the manufacturer’s instructions. Absorbance was measured with a microplate reader (SH-9000Lab; Corona Electric Co., Ltd., Ibaraki, Japan). The minimum detectable dose of IL-6 was less than 0.70 pg/mL and that for sTF was 0.16 pg/mL.
8
PC12 Cell Neuroprotection Assay
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Rat adrenal pheochromocytoma, PC12 cells, were obtained from the JCRB Cell Bank (Osaka, Japan). PC12 cells were maintained in dulbecco’s modified eagle medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin, as was described in our previous report [33 (link)]. Cells were cultured at 37 °C in humidified air containing 5% CO2 with CO2 incubator. PC12 cells were plated at 1.2 × 104 cells in poly-D-lysine-coated 96-well plates (3860-096, AGC TECHNO GLASS, Shizuoka, Japan) and incubated for 24 h. After incubation, cells were treated with 50 ng/mL NGF for 24 h for neuronal differentiation. After this step, cells were cultured in a medium without FBS but containing 50 ng/mL NGF. Cells were treated with extracts and 25 μM Aβ42 for 24 h by replacing the medium with 1% EtOH/1% DMSO. Cells were then treated with 0.5 mM MTT for 4 h by medium replacement in reference to the MTT assay [34 (link)]. After incubation, the supernatant of each well was removed, and formazan crystals were dissolved in a 10% SDS/0.01 M HCl solution. After overnight incubation, absorbance (570 nm) was measured in each well with (SH-9000Lab, CORONA ELECTRIC, Ibaraki, Japan). Cell viability was calculated as a percentage relative to cells treated with the control medium (1% EtOH/1% DMSO).
9
Cell Viability Assay Protocol for MRC-5 Fibroblasts
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Human fibroblast cells, MRC-5, were cultured on 96-well flat bottom plates at a density of 1.5 × 104 cells/well with 100 μl of MEM medium (Life Technologie) containing 10% fetal bovine serum (Hana-nesco Bio, Tokyo, Japan) and 1% penicillin-streptomycin (Life Technologies). Cells were incubated at 37 °C with 5% CO2 for 48 h. Approximately 100 μl of MEM medium containing 5 μl of each compound dissolved in 50% DMSO in water were added to each well. After the plates were incubated for 48 h, approximately 10 μl of WST-8 (Dojindo, Kumamoto, Japan) was added to each well and the plate was incubated at 37 °C with 5% CO2 for 2 h. The absorbance was measured at 450 nm by spectrophotometer (SH-9000Lab, Corona Electric, Ibaraki, Japan). Growth of MRC-5 cells was measured in duplicate. Staurosporine (Kitasato Natural Products Library) was used as a positive (cytotoxic) control in a concentration range of 0.5 pM to 0.1 nM IC50 values were calculated as described previously.
10
Quantifying Embryonic ROS Levels
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The levels of ROS in control and Pb-exposed embryos/larvae were measured using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) method. At 24, 48, and 72 hpf, the embryos whose chorion was removed and/or larvae were placed into 6-well plates filled with 10 ml of the egg culture water containing DCFH-DA at a final concentration of 20 μg/ml and incubated at 28.5°C for 30 min in dark. The embryos were then rinsed twice with egg culture water and lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 1 mM ethylenediamine-N′,N′,N′,N′-tetraacetic acid). The fluorescence of the lysates was measured (excitation at 480 nm, emission at 530 nm) using a microplate reader SH-9000Lab (CORONA ELECTRIC, Ibaraki, Japan). The ROS measurement was repeated three times for each time point using independently collected embryos from different mating groups, and three pools of embryos/larvae from the same mating group, which consisted of 13–23 embryos/larvae, were used in each trial. The results of each measurement are represented as the mean of the data from these three pools.
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