GW4064, (Z)-guggulsterone (GS), T0901317, 1α,25-vitamin D3, GW7647, and GW1929 were obtained from Cayman Chemical (Ann Arbor, MI, USA). CDCA, lithocholic acid, and Fast-blue BB salt were purchased from Sigma (St. Louis, MO, USA). GW501516 was from ChemScene LLC (Newark, NJ, USA). All-trans retinoic acid and 9-cis-retinoic acid were from FUJIFILM Wako Pure Chemical (Osaka, Japan). BMP-2 was from Miltenyi Biotec. (Bergisch Gladbach, Germany). Naphthol AS-MX phosphate was from Nacalai Tesque (Kyoto, Japan). Amino acid derivative and the coupling reagents were from Watanabe Chemical (Hiroshima, Japan) or Peptide Institute (Osaka, Japan). All other chemicals were from Tokyo Chemical Industry (Tokyo, Japan), FUJIFILM Wako Pure Chemical, and Aurora Fine Chemicals (San Diego, CA, USA) unless otherwise stated.
Naphthol as mx phosphate
Manufactured by Nacalai Tesque
Sourced in Japan
Naphthol AS-MX phosphate is a laboratory reagent used in histological and cytological applications. It functions as a substrate for the detection of phosphatase enzyme activity.
Automatically generated - may contain errors
Lab products found in correlation
Gw1929, by Cayman Chemical (1 mentions)
Bmp 2, by Miltenyi Biotec (1 mentions)
9 cis retinoic acid, by Fujifilm (1 mentions)
Gw4064, by Cayman Chemical (1 mentions)
Gw7647, by Cayman Chemical (1 mentions)
Fast blue bb salt, by Merck Group (1 mentions)
Lithocholic acid, by Merck Group (1 mentions)
Acetic acid, by Fujifilm (1 mentions)
N n dimethylformamide (dmf), by Fujifilm (1 mentions)
Sodium tartrate dehydrate, by Nacalai Tesque (1 mentions)
Fast red violet lb salt, by Merck Group (1 mentions)
4 protocols using naphthol as mx phosphate
1
Assay for Nuclear Receptor Activation
2
Osteoclast Fixation and Staining
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Adherent cells were fixed in 10% formalin for 3 min, followed by further fixation in a fixative buffer consisting of 50% ethanol and 50% acetone for 1 min. The cells were stained with Naphthol AS-MX phosphate (Nacalaitesque) and Fast-Red (Nacalaitesque) for 10–15 min at room temperature and washed with water three times. TRAP-positive cells with >3 or 10 nuclei were counted as multinucleated osteoclasts.
3
Alkaline Phosphatase Activity Assay
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Cells were seeded in a 6-well culture plate coated with recombinant laminin-511 E8 fragments at 1.3 × 104 cells/well and cultured for 5 days to induce colony formation. After washing with phosphate buffered saline (PBS), the colonies were fixed with 4% paraformaldehyde in PBS for 3 min at room temperature and exposed to a solution containing naphthol AS-MX phosphate (Nacalai Tesque) as a substrate and Fast red TR salt hemi (zinc chloride) salt (Santa Cruz Biotechnology, Dallas, TX) as a coupler for 30 min at 37 °C. AP-positive colonies were determined as the percentage of positive colonies to total colonies counted in five images of each sample under the BZ-9000 microscope.
4
TRAP Staining and Osteoclast Quantification
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TRAP staining was performed using 0.23 mM naphthol AS-MX phosphate (Nacalai Tesque Inc.) and 1.3 mM fast red violet LB salt (Sigma-Aldrich), which were dissolved by N,N-dimethylformamide (FUJIFILM Wako Pure Chemical Corporation), and then mixed with 50 mM tartrate-containing buffer (pH 5.0). The tartrate-containing buffer was composed of sodium acetate, 50 mM sodium tartrate dehydrate (Nacalai Tesque Inc.), and 1% acetic acid (FUJIFILM Wako Pure Chemical Corporation). Samples were fixed with 4% paraformaldehyde on ice for 10 min, stained with TRAP staining reagent, and incubated at 37 °C for 1 h. The TRAP-stained cells were observed using a microscope (Nikon ECLIPSE Ts2). Image analysis was performed with NIS-Elements Analysis Software (Nikon Solutions Co., Ltd., Tokyo, Japan). The numbers of TRAP-positive mononuclear osteoclasts (nuclei/cell = 1) and mature osteoclasts (nuclei/cell ≥ 3 or 10) were counted and calculated by averaging the two 96-well field of views from three independent experiments.
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