MAPC or MSC were cultured in control medium or osteogenic medium for 8 days. Alkaline phosphatase (ALP) staining was performed using a napthol AS-MX phosphate and fast red violet B salt-based kit (Sigma–Aldrich). Briefly, cells were fixed in citrate-buffered acetone and rinsed with water. Cells were exposed to the alkaline dye mixture for 30 min, rinsed with water, visualized using light microscopy, and imaged. Images were taken at 10× magnification.
Napthol as mx phosphate
Manufactured by Merck Group
Sourced in United Kingdom, United States
Napthol AS-MX phosphate is a laboratory reagent used as a substrate for the detection of various enzymes, particularly alkaline phosphatase. It is a colorless compound that, when cleaved by the target enzyme, produces a colored compound that can be visualized.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Fast red tr, by Merck Group (2 mentions)
N n dimethylformamide (dmf), by Merck Group (2 mentions)
Fast red violet lb salt, by Merck Group (2 mentions)
Fast red tr salt, by Merck Group (2 mentions)
Sodium acetate, by Merck Group (2 mentions)
Tartaric acid, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Rankl, by Miltenyi Biotec (1 mentions)
Dimethylformamide, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
M csf, by Miltenyi Biotec (1 mentions)
Lympholyte m, by Cedarlane (1 mentions)
Cavin1, by Proteintech (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Geneticin g418, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (1 mentions)
α minimum essential medium α mem, by Thermo Fisher Scientific (1 mentions)
Cellvue claret far red fluorescent cell linker kit, by Merck Group (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
8 protocols using napthol as mx phosphate
1
Osteogenic Differentiation Assay
2
Fluorescent Staining for Cell Analysis
3
Fast Red Staining Protocol
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Cells were washed with PBS, fixed in 100% methanol for 10 min and air dried. Then, cells were incubated with the staining solution (mixture of 2 mg ml−1 Napthol AS-MX phosphate (Sigma) in 0.1 M Tris–HCl pH 9.2 and 1 mg ml−1 Fast Red TR saltTM (Sigma) in 0.1 M Tris–HCl pH 9.2 to 1:10 dilution) for 10–15 min in dark. The reaction was stopped reaction by rinsing cells twice with distilled water.
4
Osteoclast Differentiation from Mouse Bone Marrow
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Bone marrow (BM) cells isolated from 12 weeks old C57BL/6J and CD1 mice were density separated with Lympholyte-M (Cedarlane, Burlington, USA) and the purified lymphocytes were cultured overnight at 37 °C in 5% CO2 in complete medium: α-MEM with 10% foetal bovine serum (FBS), supplemented with 50 ng/ml murine macrophage colony stimulating factor (M-CSF) (Miltenyi, Abingdon, UK) and next day were plated at a density of 1 × 10^5 cells/well in 96-well plates where OC precursors were induced to proliferate and differentiate with (150 ng/ml) M-CSF and (10–100 ng/ml) receptor activator of NF-kB ligand (RANKL) (Miltenyi, Abingdon, UK) supplemented with 0–10^4 nM Dexamethasone (Dex) (Sigma-Aldrich, Gillingham, UK) as indicated. On day 6 cells were washed with PBS and fixed with 4% paraformaldehyde (PFA), then permeabilized with acetone/ethanol 1:1 solution and stained for TRAP activity for 15 mins at 37 °C (0.1 mg/ml Napthol AS-MX phosphate, 0.4 mg/ml Fast Red Violet LB Salt, 0.5 ml Dimethylformamide in 50 ml TRAP buffer; all from Sigma-Aldrich, Haverhill Suffolk, UK). Multinucleated (no. of nuclei ≥3) TRAP-positive cells that stained red were considered to be OCs. In all the experiments BM cells were also cultured in complete medium with M-CSF only served as negative control for OC formation.
5
Immunofluorescence Labeling of Cellular Markers
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Reagents and antibodies used were provided by the following sources: Roswell Park Memorial Institute (RPMI)-1640 medium, α-Minimum Essential Medium (α-MEM), Dulbecco's Modified Eagle Medium (DMEM), L-glutamine, HEPES, Geneticin (G418), penicillin–streptomycin, ProLong Gold Antifade and trypsin were from Life Technologies (Grand Island, NY, USA). Fetal bovine serum (FBS) was from Thermo Scientific (Mordialloc, Vic, AUS). Fast Red Violet LB Salt, napthol AS-MX Phosphate, PKH2 and CellVue Claret Far Red Fluorescent Cell Linker Kits were from Sigma-Aldrich (Saint Louis, MO, USA). Antibodies against calnexin and caveolin-1 were from BD Transduction Laboratories (Franklin Lakes, NJ, USA), cofilin and EphA2 from Cell Signalling Technology (Franklin Lakes, NJ, USA), cavin-1 from ProteinTech (Chicago, IL, USA), α-tubulin and 4F2 from Sigma-Aldrich (Saint Louis, MO, USA), and CD63 from Developmental Studies Hybridoma Bank (Iowa City, IA, USA).
6
Alkaline Phosphatase Staining of Porcine ICM Cells
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Cultured porcine ICM-derived cells were fixed in 4% (v/v) paraformaldehyde (Junsei Chemical Co., Japan). After two washes with Dulbecco’s phosphate-buffered saline (DPBS; Welgene), the fixed cells were stained with a solution containing 0.2 mg/ml napthol AS-MX phosphate (Sigma-Aldrich), 2% (v/v) N,N-dimethylformamide (Sigma-Aldrich), and 1 mg/ml Fast Red TR salt (Sigma-Aldrich) in 0.1 M Tris buffer (pH 8.2) for 90 min at room temperature. Subsequently, the stained cells were rinsed twice with DPBS, and the proportion of AP-positive cells was measured using a hemocytometer and an inverted microscope (CKX-41; Olympus, Japan).
7
Quantitative Analysis of Osteoclast Formation
8
Quantifying TRAP+ Osteoclasts in Tissue
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To measure TRAP+ osteoclasts, paraffin sections were deparaffinized and incubated in acetate-tartaric acid buffer (0.2 M sodium acetate [Merck, 6268], 50 mM tartaric acid [Sigma-Aldrich, T10-9], pH 5.0) for 20 min at 37°C and then stained in 1.1 mg/mL FAST Red TR (Sigma, F8764-16) substrate and 0.1−0.5 mg/mL napthol ASMX phosphate (Sigma, N4875) substrate. Sections were incubated for 2−4 h until sufficient red color was observed in osteoclasts, washed in PBS, and then counterstained with Methyl Green (Vector Laboratories, H-3402-500) before washing, air drying, and mounting in VectaMount permanent mounting medium (Vector Laboratories, H-50000). Masson trichrome staining was performed using the Masson Trichrome Stain Kit (SKU KTMTRLT EA, StatLab, TX, USA) according to the manufacturer’s instructions. Images were obtained using a Zeiss Colibri inverted microscope at 10x and 20x dry magnification using a Zeiss 305 color camera.
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