After washing with DPBS, the cells were fixed in 4% paraformaldehyde (16005, Sigma) and rinsed with deionized water thoroughly. Cells were stained with Alizarin Red S (A5533, Sigma) solution (2%, pH 4.2) for 20 min at room temperature. Excessive dye was removed by several washes in deionized water. To quantify AR staining in 96-well plates, we added 100 μL of hexadecylpyridinium chloride (C9002, Sigma) solution (100 mmol/L) to the wells and measured optical density (OD) at 560 nm using hexadecylpyridinium chloride solution as blank.
Hexadecylpyridinium chloride
Manufactured by Merck Group
Sourced in United States
Hexadecylpyridinium chloride is a quaternary ammonium compound used as a surfactant and biocide in various applications. It has a chemical formula of C21H38ClN.
Automatically generated - may contain errors
Lab products found in correlation
Spectramax 190, by Molecular Devices (3 mentions)
Alizarin red, by Solarbio (3 mentions)
Ars solution, by Merck Group (2 mentions)
Alizarin red s, by Merck Group (1 mentions)
Streptomycin sulfate, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Alizarin red s solution, by Cyagen (1 mentions)
Sodium β glycerophosphate, by Merck Group (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Cell counting kit 8 cck 8, by Keygen Biotech (1 mentions)
Ars solution, by Solarbio (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Formaldehyde, by Solarbio (1 mentions)
Alizarin red s solution, by Solarbio (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
L wenstein jensen medium, by BD (1 mentions)
Middlebrook 7h11 medium, by BD (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
16 protocols using hexadecylpyridinium chloride
1
Quantification of Alizarin Red S Staining
2
Optimizing Bone Cell Culture Conditions
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6-aminocaproic acid, L-hydroxyproline, L-alanine, L-phenylalanine, L-proline and L-lysine (99.0%, biochemical grade) were purchased from Hebei kairuijie amino co., Ltd. (Xintai, China). The pearl powder with 0.5–10 µm diameter was purchased from STS Biotech Co., Ltd. (Wuxi, China). Minimum essential medium eagle-alpha modification (α-MEM), fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin sulfate were purchased from Gibco (Grand Island, NY, USA); Cell counting kit-8 (CCK-8) was purchased from Nanjing Keygen Biotech Co., Ltd. (Nanjing, China); Glutaraldehyde (50.0%, AR) and formaldehyde (37.0–40.0%, AR) were purchased from Chengdu Chron Chemicals Co., Ltd. (Chengdu, China); Rhodamine-phalloidin was obtained from Servicebio Co., Ltd. (Wuhan, China), Dexamethasone (98.0%), ascorbic acid (99.0%), β-glycerophosphate sodium (99.0%) and hexadecylpyridinium chloride (99.0–102.0%) were purchased from Sigma-Aldrich Co., Ltd. (Saint Louis, MO, USA); Mouse Alkaline Phosphatase Activity (ALP) Enzyme-linked Immunosorbent Assay (ELISA) Kit was purchased from Shanghai MLBIO Biotechnology Co., Ltd., Alizarin Red S solution was purchased from Cyagen Biosciences Co., Ltd. (Guangzhou, China), and TRIZOL reagent from Ambion (Carlsbad, CA, USA).
3
Osteogenic Differentiation of hDPSCs
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hDPSCs were seeded into a 12-well plate and cultured with osteogenic medium for 21 days. After being fixed with 4% formaldehyde (Solarbio) for 30 minutes, cells were stained with 2% ARS solution (Solarbio) at 37 °C for 10 minutes, followed by eluting with hexadecylpyridinium chloride (100 mM, Sigma-Aldrich) for 1 hour. Later, the absorbance was detected at 562 nm by a microplate reader (Bio-Rad).
4
Alizarin Red S Staining for Mineralized Nodules
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Alizarin Red S staining was used to estimate the mineralized nodule formation after 21 d of incubation. The cells on each sample were fixed by 4% paraformaldehyde for 20 min, and 1% Alizarin Red S solution (pH = 4.2, Solarbio, China) was added to each well for 20 min. The excess solution was then thoroughly removed with deionized water, and the deposited calcium was recorded. For further quantification, 10% hexadecylpyridinium chloride (w/v, Sigma) was added to dissolve the red matrix sediment completely, and 100 μL solution per well was transferred into a 96-well plate and measured by a microplate reader at a wavelength of 550 nm.
5
Mycobacterium avium subsp. paratuberculosis Culture
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Culture methodology was performed as described by Juste et al.,1991 [15 (link)] and Aduriz et
al., 1995 [1 (link)]. Samples from all animals were decontaminated with 0.75% (w/v) hexadecyl pyridinium chloride
(HPC; Sigma-Aldrich, Milano, Italy) for 18 hr and cultured, in duplicate, using five specific media, supplemented with a mix of amphotericin B (50
mg/l), penicillin (100,000 U/l) and chloramphenicol (100 mg/l) (Sigma-Aldrich).
The five media used in this study were Löwenstein-Jensen medium (Liofilchem, Roseto degli Abruzzi, Italy), Löwenstein-Jensen medium with mycobactin J
(Synbiotics Europe SAS, Lyon, France), Löwenstein-Jensen medium with sodium pyruvate without glycerol, Middlebrook 7H11 medium supplemented
with OADC (oleic acid-albumin-dextrose-catalase) (Becton Dickinson, Franklin Lakes, NJ, U.S.A.) and Middlebrook 7H11 medium supplemented with OADC and sodium
pyruvate without glycerol. All culture media were incubated at 37°C for 34–52 weeks and checked every week for mycobacterial growth or contamination with
undesirable microorganisms. Colonies with compatible mycobacterial morphology were tested for acid fastness bacilli by the Ziehl-Neelsen stain of smears method.
The mycobacterial isolates were tested for Map confirmation by the PCR methods described above.
al., 1995 [1 (link)]. Samples from all animals were decontaminated with 0.75% (w/v) hexadecyl pyridinium chloride
(HPC; Sigma-Aldrich, Milano, Italy) for 18 hr and cultured, in duplicate, using five specific media, supplemented with a mix of amphotericin B (50
mg/l), penicillin (100,000 U/l) and chloramphenicol (100 mg/l) (Sigma-Aldrich).
The five media used in this study were Löwenstein-Jensen medium (Liofilchem, Roseto degli Abruzzi, Italy), Löwenstein-Jensen medium with mycobactin J
(Synbiotics Europe SAS, Lyon, France), Löwenstein-Jensen medium with sodium pyruvate without glycerol, Middlebrook 7H11 medium supplemented
with OADC (oleic acid-albumin-dextrose-catalase) (Becton Dickinson, Franklin Lakes, NJ, U.S.A.) and Middlebrook 7H11 medium supplemented with OADC and sodium
pyruvate without glycerol. All culture media were incubated at 37°C for 34–52 weeks and checked every week for mycobacterial growth or contamination with
undesirable microorganisms. Colonies with compatible mycobacterial morphology were tested for acid fastness bacilli by the Ziehl-Neelsen stain of smears method.
The mycobacterial isolates were tested for Map confirmation by the PCR methods described above.
6
Alizarin Red Staining of Vascular Smooth Muscle Cells
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After cultured at specified conditions for 14 days, VSMCs on dishes were fixed with 10% formalin for 30 min, then washed with double distilled water (ddH2O) twice and incubated with Alizarin Red (Solarbio Life Sciences, China) for 5 min and washed with ddH2O twice to remove the excessive dye. After examination and photography under a microscopy, the dye on the cells was extracted with 100ul Hexadecyl Pyridinium chloride (Sigma-Aldrich) and the optical density (OD) at 560 nm was measured using a microplate reader (Spectra MAX 190, Molecular Devices, USA).
7
Alizarin Red-Based Mineralization Quantification
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Alizarin red staining and measurement were performed to measure mineralization. Cultured cells were fixed with ice-cold ethanol 96% for 30 min in a 24-well plate. After washing twice with dH2O, 400 μl alizarin red staining solution was added in each well for 10 min, washed again four times. The stain was eluted for 20 min into a solution of 10% hexadecylpyridiniumchloride (Sigma-Aldrich). Absorbance was read at 562 nm. Alizarin red concentration for samples was calculated according to standard curve and results were normalized to the protein content analysed via SRB staining [23 (link)].
8
Chromium Speciation Analysis Protocol
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Pure water (18 MΩ·cm resistivity) obtained from a Milli-Q system (Millipore, Belford, MA, USA) was used in the preparation of the solutions. Plastic and glassware were washed with diluted nitric acid (1% v/v concentrated acid) before use. Standard Cr3+ and Cr6+ (1 g L−1) solutions were prepared from solid Cr(NO3)3·9H2O and K2Cr2O7 (Fluka, Buchs, SG, Switzerland), respectively, and diluted conveniently daily to prepare working solutions. The reagent 1,5-diphenylcarbazide, DPC, (0.05 mol L−1) was prepared by weighing the required amount of product (Sigma-Aldrich, Steinheim, Germany) and dissolving it in acetone. The surfactants sodium dodecylsulfate (SDS), hexadecylpyridinium chloride, hexadecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, IGEPAL®, and Triton X-45, X-100, and X-114 and the rest of the reagents used were obtained from Sigma-Aldrich.
9
Alizarin Red Staining for ECM Mineralization
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Alizarin red staining (ARS) was used to assess the ECM mineralization of the cells cultured on different samples. After 7 days and 14 days of culture, the cell-seeded samples were fixed in 4% paraformaldehyde for 30 min. Then, the samples were stained with ARS solution (2%, pH 4.2; Sigma-Aldrich) for 20 min. After washing the stained samples with deionized water several times to remove the excess ARS, the stained pictures of the samples were taken with an optical microscope. To quantitatively reveal the ECM mineralization of the samples, the stained samples were also immersed into hexadecyl pyridinium chloride (1 w/v%; Sigma-Aldrich) and shaken for 2 h. The absorbance values were measured at 550 nm. Three samples for each group were tested, and each test was repeated three times (n = 3).
10
Alizarin Red Staining of Calcified SMCs
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After cultured at specified conditions for 14 days, SMCs on dishes were fixed with 10% formalin for 30 min, then washed with double distilled water (ddH2O) twice and incubated with Alizarin Red (Solarbio Life Sciences, China) for five minutes and washed with ddH2O twice to remove the excessive dye. After examination and photography under a microscopy, the dye on the cells was extracted with 100µl Hexadecyl Pyridinium chloride (Sigma-Aldrich) and the OD at 560 nm was measured using a microplate reader (Spectra MAX 190, Molecular Devices, USA).
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