To examine the effects of these test ingredients on osteogenic differentiation, MC3T3-E1 cells were washed and seeded onto 24-well plates and incubated in complete α-MEM for 24 h for cell adherence and growth. Then, the medium was replaced with the osteoblast inducing conditional media (complete α-MEM supplemented with 1% β-glycerophosphate and 1% ascorbic acid). At the same time, the selected ingredients were added into the osteogenic medium at three concentration gradients (three repeat wells per concentration). The cultures were maintained at 37 °C with 5% CO2, and the medium was replaced every two days. ALP staining was monitored using a BCIP/NBT Alkaline Phosphatase Color Development Kit. Cells were fixed by immersion 4% paraformaldehyde (PFA) solution for 10 min and rinsed in PBS for 5 min 3 times. The samples were then placed in an alkaline phosphatase staining solution (BCIP/NBT solution) for 30 min. The whole procedure was protected from light. After discarding the solution, cells were rinsed in deionized water 2 min 2 times and scanned with a CanoScan 9000F Mark II scanner (Canon, Tokyo, Japan). Alizarin red staining of mineralized osteoblast nodules was carried out. Briefly, cells were fixed and washed as above and then stained in 0.5% Alizarin red S (pH 4.0) for 30 min. After washing, the plates were scanned with a CanoScan 9000F Mark II scanner.
Canoscan 9000f mark 2 scanner
Manufactured by Canon
Sourced in Japan, China
The CanoScan 9000F Mark II scanner is a high-performance flatbed scanner designed for professional-grade image digitization. It features a maximum optical resolution of 9600 x 9600 dpi, allowing for the capture of fine details and high-quality scans. The scanner supports a wide range of media, including film, slides, and reflective documents.
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Lab products found in correlation
Alizarin red s, by Merck Group (2 mentions)
Bcip nbt alkaline phosphatase color development kit, by Beyotime (2 mentions)
Paraformaldehyde (pfa), by Wuhan Servicebio Technology (2 mentions)
Crystal violet, by Solarbio (2 mentions)
Cck 8 reagent, by MedChemExpress (2 mentions)
Westernbright quantum hrp substrate, by Advansta (1 mentions)
Hrp coupled secondary antibodies, by Southern Biotech (1 mentions)
Fusion fx7, by Vilber (1 mentions)
Alcian blue, by Merck Group (1 mentions)
Alizarin red staining solution, by Merck Group (1 mentions)
Alizarin red, by Merck Group (1 mentions)
Paraformaldehyde pfa, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Protease inhibitor cocktail, by Solarbio (1 mentions)
Collagenase, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
P nitrophenyl phosphate substrate, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Cytiva (1 mentions)
11 protocols using canoscan 9000f mark 2 scanner
1
Osteogenic Differentiation of MC3T3-E1 Cells
2
Western Blot Protocol for Protein Analysis
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Proteins were separated on 10% Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto nitrocellulose membranes with semi-dry or wet transfer systems (BioRad, Hercules, CA, USA), depending on the size of proteins to be analyzed. The membranes were stained with Ponceau red protein stain for 15 min, rinsed with ddH2O and scanned with a CanoScan 9000F Mark II scanner (Canon). The Ponceau red was washed off and the membrane blocked with 5% milk in PBS-Tween20 for 1 h at RT or overnight at 4 °C. The antibodies were diluted in 5% milk PBS-Tween20 at 1:15,000 for anti-coronin A, and 1:5000 for anti-actin. Primary antibody incubation was done either at room temperature for 2 h or overnight at 4 °C, followed by washes and by incubation with horseradish peroxidase (HRP)-coupled secondary antibodies (Southern Biotech). Membranes were developed using SuperSignal PicoWest chemiluminescence substrate (Thermo-Fisher) or WesternBright Quantum HRP substrate (Advansta) and imaged using a Fuji FPM 800A (Fuji, Tokyo, Japan) or Fusion FX7 (VILBER, Paris, France)
3
Skeletal Analysis of Postnatal Mice
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Skeletal structures of mice at postnatal day 0 were analysed by whole‐mount Alizarin red/Alcian blue staining. All mice were skinned and eviscerated, and fixed in 95% ethanol overnight. Fat tissues were removed with acetone at room temperature. Cartilage tissue of mice was stained with Alcian blue (Sigma) for 24 hours. After rinsed in 70% ethanol for 8 hours, soft tissues were removed with 1% KOH. Bones were counterstained with Alizarin red (Sigma) overnight and subsequently cleared with 1% KOH/20% glycerol for 2 days or more at 4°C. Samples were stored in glycerol:ethanol (1:1) until imaging. The width of cranial suture, mineralized region rate of 9th rib and mineralized region rate of femur were measured by Image J analysis software (Image J).
For Alizarin red S (ARS) staining of cells, osteoblasts after differentiation in 24‐well plate were washed with PBS, fixed with 4% Paraformaldehyde (PFA) at room temperature for 15 minutes and stained with 0.5% Alizarin red staining solution (pH = 4.0) (Sigma) for 20 minutes at room temperature. Then, the cells were washed with tap water for 3‐5 times. The mineralized nodules were scanned by a CanoScan 9000F Mark II scanner (Canon).
For Alizarin red S (ARS) staining of cells, osteoblasts after differentiation in 24‐well plate were washed with PBS, fixed with 4% Paraformaldehyde (PFA) at room temperature for 15 minutes and stained with 0.5% Alizarin red staining solution (pH = 4.0) (Sigma) for 20 minutes at room temperature. Then, the cells were washed with tap water for 3‐5 times. The mineralized nodules were scanned by a CanoScan 9000F Mark II scanner (Canon).
4
Osteogenic Differentiation Analysis via ALP and Alizarin Red Staining
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ALP staining and Alizarin Red Staining were performed to determine osteogenic differentiation. Alkaline phosphatase (ALP) of MC3T3‐E1 cells and BMSCs was stained using BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime Biotechnology, C3206, Shanghai, China) according to the manufacturer's instruction. Briefly, cells were washed with PBS (pH 7.4) and fixed in 10% buffered formaldehyde. The formaldehyde was washed by PBS, and then, cells were stained using 5‐bromo‐4‐chloro‐3‐indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) solution. The staining reaction was stopped by washing with tap water, and the cell staining was imaged by CanoScan 9000F Mark II Scanner (Canon, Tokyo, Japan).
For Alizarin Red Staining, cells were cultured in osteogenic medium and the medium was replaced every 2 days. The staining was carried out on day 22 for MC3T3‐E1 cells and day 26 for BMSCs, and cells were washed with distilled water and then stained in 0.5% Alizarin Red S (pH 4.0; Sigma, A5533) for 30 mins. After immersed by tap water for 30 mins, the plates were dried and scanned with CanoScan 9000F Mark II Scanner.
For Alizarin Red Staining, cells were cultured in osteogenic medium and the medium was replaced every 2 days. The staining was carried out on day 22 for MC3T3‐E1 cells and day 26 for BMSCs, and cells were washed with distilled water and then stained in 0.5% Alizarin Red S (pH 4.0; Sigma, A5533) for 30 mins. After immersed by tap water for 30 mins, the plates were dried and scanned with CanoScan 9000F Mark II Scanner.
5
Proliferation and Colony Formation Assay for Transfected GC Cells
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Transfected GC cells (2 × 10 3 /well) were cultured in 96-well plates, then incubated with 10 μL of the CCK-8 reagent (MedChemExpress, Monmouth Junction, NJ, USA) for 2 h. Absorbance at 450 nm was detected at 0, 24, 48, and 72 h thereafter.
Transfected GC cells (6 × 102/well) were cultured in 6-well plates for 8 d, fixed in paraformaldehyde (Servicebio, Wuhan, China), and stained with crystal violet (Solarbio Co., Ltd., Beijing, China). Thereafter, colony formation was assayed using a CanoScan 9000F Mark II scanner (Canon Inc., Tokyo, Japan).
Transfected GC cells (6 × 102/well) were cultured in 6-well plates for 8 d, fixed in paraformaldehyde (Servicebio, Wuhan, China), and stained with crystal violet (Solarbio Co., Ltd., Beijing, China). Thereafter, colony formation was assayed using a CanoScan 9000F Mark II scanner (Canon Inc., Tokyo, Japan).
6
Quantification of M-CSF Protein in Breast Cancer Cells
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The breast cancer cells were lysed in RIPA lysis buffer (Solarbio, Peking, China) containing a protease inhibitor cocktail (Solarbio, Peking, China). The total protein in the supernatant was quantified using a bicinchoninic acid (BCA) protein assay kit (Beyotime, China). The proteins were extracted and resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 10% gel. The proteins were then transferred to a polyvinylidene fluoride membrane. The membrane was incubated in a solution containing primary antibodies against M-CSF and GAPDH (Proteintech, Wuhan, China) overnight at 4 °C. The protein bands were visualized using radiographic film exposure. The blots were documented using Canon CanoScan 9000F Mark II scanner (Canon, Peking, China).
7
Gelatin Degradation Assay for Macrophage-Conditioned Media
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The CM of macrophages was analyzed for gelatin degradation by electrophoresis using sodium dodecyl-polyacrylamide gel containing 1 mg/mL gelatin. The volume of CM loaded per lane was standardized on the basis of the cell count. The gels were incubated overnight at room temperature (25 °C) in 50 mm Tris-HCl, 150 mm NaCl, and 10 mm CaCl2 (pH 7.4). The white lysis zone, which indicated the gelatin degradation, was identified by staining with Coomassie brilliant blue. The blots were documented using Canon CanoScan 9000F Mark II scanner (Canon, Peking, China).
8
Isolation and Differentiation of Primary Osteoblast Precursors
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Primary osteoblast precursors were isolated from the calvarias of neonatal ICR mice via enzymatic digestion with 0.1% collagenase (Life Technologies, Carlsbad, CA, USA) and 0.2% dispase II (Roche Diagnostics, GmbH, Mannheim, Germany). Primary osteoblast precursors were differentiated into osteoblasts by culturing in α-MEM containing 10% FBS (HyClone Laboratories, Logan, UT, USA), 100 U/mL penicillin, 100 mg/mL streptomycin (Life Technologies, Carlsbad, CA, USA), BMP2 (100 ng/mL), ascorbic acid (50 µg/mL), and β-glycerophosphate (100 mM). The cultured cells were lysed in 50 mM Tris-HCl (pH 7.4) containing 1% Triton X-100, 150 mM NaCl, and 1 mM EDTA and incubated with p-nitrophenyl phosphate substrate (Sigma-Aldrich, St. Louis, MO, USA). ALP activity was then assessed by measuring the absorbance at 405 nm using a spectrophotometer. Cultured cells were fixed with 4% paraformaldehyde, stained with 40 mM alizarin red (pH 4.2), and washed with phosphate-buffered saline (PBS) to remove nonspecific staining. Then, the cells were visualized using the CanoScan 9000F Mark II scanner (Canon Inc., Tokyo, Japan). The quantitation of alizarin red was performed by extracting alizarin red from the stained cells with 10% acetic acid for 30 min. Subsequently, the absorbance of the samples was measured at 405 nm via spectrophotometry.
9
Osteoblast Differentiation Assessment via Staining
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ALP staining and Alizarin red Staining were performed to determine osteoblast differentiation. Alkaline phosphatase (ALP) of osteoblasts was stained by BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime Biotechnology, C3206, Shanghai, China) according to the manufacturer’s instruction. Briefly, cells were washed with PBS (pH7.4) and fixed in 10% buffered formaldehyde. The formaldehyde were washed and then cells were stained using 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) solution. The staining reaction was stopped by washing with distilled water and the cell staining was imaged by CanoScan 9000F Mark II scanner (Canon, Tokyo, Japan) and Relative Staining Area of ALP was analyzed by Image-Pro Plus 6.0 software.
For Alizarin red Staining, cells were cultured in osteogenic medium. The staining was carried out on day 25, cells were washed with PBS and then stained in 0.5% Alizarin red S (PH 4.0, Sigma, A5533) for 30mins. After washes with tap water, the plates were dried and scanned with CanoScan 9000F Mark II scanner and Relative Staining Area of Alizarin red S was analyzed by Image-Pro Plus 6.0 software.
For Alizarin red Staining, cells were cultured in osteogenic medium. The staining was carried out on day 25, cells were washed with PBS and then stained in 0.5% Alizarin red S (PH 4.0, Sigma, A5533) for 30mins. After washes with tap water, the plates were dried and scanned with CanoScan 9000F Mark II scanner and Relative Staining Area of Alizarin red S was analyzed by Image-Pro Plus 6.0 software.
10
Cell Proliferation and Colony Formation
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Transfected GC cells (2 × 103 cells/well) were cultured in 96-well plates for 0, 24, 48, and 72 h and were then incubated with 10 μL of CCK-8 reagent (MedChemExpress, Monmouth Junction, NJ, USA) for 2 h. The absorbance was measured at 450 nm. Transfected GC cells (6 × 102 cells/well) were cultured in 6-well plates for 8 d, fixed with paraformaldehyde (Servicebio, Wuhan, China), and stained with crystal violet (Solarbio Co. Ltd., Beijing, China). Then, colony formation was assayed using a CanoScan 9000F Mark II scanner (Canon Inc., Tokyo, Japan).
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