Alkaline phosphatase staining was carried out as described previously.44 (link) The cells were seeded in 6-well plates at a density of 2 × 104 cells per well. After in vitro osteogenic induction for 7, 14, and 21 days performed as previously described, alkaline phosphatase staining was carried out. Briefly, culture wells were rinsed three times with PBS, fixed in 95% alcohol for 1 min, washed three times with PBS, incubated with the substrate mixture (2% sodium β-glycerophosphate, 25 ml; 2% sodium barbiturate, 25 ml; distilled water, 50 ml; 2% calcium chloride, 5 ml; 2% magnesium sulfate, 2 ml; and several drops of chloroform; Solarbio, Beijing, China) at 37 °C for 2 h, washed twice with pure water, incubated in 2% cobalt nitrate (Solarbio) for 2 min, and finally incubated with 2% amine sulfide (Solarbio) for 2 min. Stained cell images were captured at the same magnification and light intensity.
Chloroform
Manufactured by Solarbio
Sourced in China
Chloroform is a colorless, dense liquid chemical compound with the chemical formula CHCl3. It is commonly used as a solvent in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Phenol, by Solarbio (2 mentions)
Sybr safe, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Isopropanol, by Sinopharm (1 mentions)
Tb green premix ex taq 2 tli rnaseh plus, by Takara Bio (1 mentions)
Primerscript rt reagent kit, by Takara Bio (1 mentions)
Dimethyl sulfoxide (dmso), by Solarbio (1 mentions)
Phosphate buffered saline (pbs), by Solarbio (1 mentions)
Rpmi 1640 medium, by Solarbio (1 mentions)
Trypan blue, by Solarbio (1 mentions)
Isopropanol, by Solarbio (1 mentions)
Diethyl pyrocarbonate treated water, by Solarbio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Mannose, by Yuanye Bio-Technology (1 mentions)
Arabinose, by Yuanye Bio-Technology (1 mentions)
Galactose, by Yuanye Bio-Technology (1 mentions)
Galacturonic acid, by Yuanye Bio-Technology (1 mentions)
Rhamnose, by Yuanye Bio-Technology (1 mentions)
Xylose, by Yuanye Bio-Technology (1 mentions)
Glucuronic acid, by Yuanye Bio-Technology (1 mentions)
7 protocols using chloroform
1
Alkaline Phosphatase Staining for Osteogenesis
2
Genomic DNA Extraction and Genotyping PCR
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Mouse tail samples (0.2–0.3 cm) were obtained and lysed at 55°C overnight in lysis buffer containing 1 mM Tris-HCl (pH 8.0), 1 mM NaCl, 1 mM EDTA (pH 8.0), 0.5% SDS and 10 µg/µl proteinase K (Sigma-Aldrich; Merck KGaA). Genomic DNA was extracted from the lysate using phenol and chloroform (Beijing Solarbio Science & Technology Co., Ltd.), precipitated with isopropanol (Sinopharm Chemical Reagent Co., Ltd.) and dissolved in distilled water at 60°C. Subsequent PCR was performed using a Taq DNA polymerase kit (cat. no. M1661S; Promega Corporation) according to the manufacturers instruction. Primers for genotyping were designed: 5′-CAGCCGATGCTTCTATGACAAC-3′ (forward), 5′-TCCCACATTTTCCGCAGGTG-3′ (reverse 1), 5′-TCTGGGCTCCGTAATGTCTG-3′ (reverse 2). The PCR conditions were as follows: Denaturation at 94°C for 5 min, followed by 35 cycles at 94°C for 50 sec, 55°C for 1 min, 65°C for 50 sec, and a final extension at 65°C for 10 min. PCR products were separated on 1.2% agarose gels by electrophoreses in 1X TBE buffer. The products were visualized using a gel imaging system (Tanon Science and Technology Co., Ltd.) after staining with SYBR Safe™ (Thermo Fisher Scientific, Inc.).
3
Purified MCP Cytotoxicity Assay
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Purified MCP (purity ≥90%) was obtained from Chenguang Biotech Group Co., Ltd. Methyl thiazole blue (MTT), trypan blue, RPMI-1640 medium, DMSO, trimethylol aminomethane, PBS and chloroform were obtained from Beijing Solarbio Science & Technology Co., Ltd. PrimerScript™ RT reagent Kit and TB Green™ Premix Ex Taq™ II (Tli RNase H Plus) kits were purchased from Takara Biotechnology Co., Ltd.
4
RNA Extraction and Reverse Transcription Protocol
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Total RNA from treated cells and frozen liver was extracted using Trizol reagent (Life Technologies, Carlsbad, CA, USA). RNA was treated with 0.2 mL chloroform (Solarbio, Beijing, China) and 0.4 mL isopropanol (Solarbio, Beijing, China), followed centrifugation at 20 °C. The pellet was resuspended in 0.5 mL diethyl pyrocarbonate-treated water (Solarbio, Beijing, China), and 2 µL was used to determine concentration. PCR was performed according to manufacturer instructions for the reverse transcription kit (Tiangen Biotechnology Co., Ltd., Beijing, China). RNA was added to a mixture of 5× g DNA buffer (Aidlab Biotechnologies Co., Ltd., Beijing, China), FQ-RT primer mix (Thermo Fisher Scientific, Waltham, MA, USA) 2 μL, 10× King RT buffer (Tiangen Biotechnology Co., Ltd., Beijing, China), and RNase-free ddH2O (Tiangen Biotechnology Co., Ltd., Beijing, China) to a final volume of 10 μL and incubated at room temperature. PCR cycling conditions were as follows: 42 °C for 15 min and 95 °C for 3 min, followed by 1 cycle of 30 °C for 10 min, 42 °C for 1 h, 95 °C for 5 min, and a final rest period at 12 °C. Primers are listed in Supplementary Table S2 .
5
Bioactive Compound Extraction from Hemerocallis citrina
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Hemerocallis citrina Borani was obtained from Qingyang, Gansu, China. Standards of glucose, mannose, fucose, rhamnose, xylose, galactose, arabinose, glucuronic acid, ribose, and galacturonic acid were purchased from Shanghai Yuanye Biotechnology (Shanghai, China). Absolute ethanol was purchased from Fuyu Fine Chemical (Tianjin, China). Trifluoroacetic acid, methanol, sodium hydroxide, hydrochloric acid, carbazole, 1-phenyl-3-methyl-5-pyrazolone, 3,5-dinitrosalicylic acid, sulfuric acid, Coomash bright blue, chloroform, potassium bromide, sodium azide, sodium acetate, anthranone, and other reagents were purchased from Solarbio Biotechnology (Beijing, China). ABTS and FRAP assay kits were purchased from Suzhou Keming Biotechnology (Suzhou, China). Distilled water was used in these experiments.
6
Multifunctional Drug Delivery Platform
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1,4,8,11,15,18,22,25-Octabutoxy-29H,31H-phthalocyanine [(BuO)8PCH2], 1,3-diphenylisobenzofuran (DPBF), and polylactic-co-glycolic acid (PLGA, 50:50) were purchased from Sigma-Aldrich (Shanghai, China). N-Hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N′-ethyl carbodiimide hydrochloride (EDC·HCl), and DTX were obtained from Sangon Biotech (Shanghai) Co., Ltd. HA (10 kDa) was bought from Rhawn Science and Technology (Shanghai, China) Co., Ltd. Manganese chloride (MnCl2), 1-hexadecanamine (HDA), and hyaluronidase (HAase) were bought from Shanghai Yuanye Bio-Technology Co., Ltd. Dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dichloromethane (DCM), ethanol, and chloroform were derived from Solarbio (Beijing, China). All reagents were of analytical grade and had no additional purification.
7
Immunoprecipitation and RNA Extraction Protocol
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The HEK293 cells were washed twice with pre-cooled PBS and lysed with an equal volume of RIPA lysis buffer (Beyotime, Shanghai, China). RIPA wash buffer (Meixuan Biological Technology Co., Ltd., Shanghai, China) was used to prepare the magnetic beads. Then, 100 μl of cell lysate was added to the magnetic beads and resuspended in 900 μl of RIPA buffer (Meixuan Biological Technology Co., Ltd., Shanghai, China), and incubated overnight at 4°C. The magnetic bead and antibody complex were resuspended with proteinase K buffer (Meixuan Biological Technology Co., Ltd., Shanghai, China), and incubated for 30 min at 55°C, and washed in RIPA buffer, phenol, and chloroform (SolarBio, Beijing, China). Finally, salt solution and precipitate enhancer were added (Meixuan Biological Technology Co., Ltd., Shanghai, China) and anhydrous ethanol was also added and incubated at 80°C for 1h. After centrifuging, the precipitate was dissolved in diethylpyrocarbonate (DEPC) (Sigma-Aldrich, St. Louis, MO, USA) for further analysis.
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