After fixation with Lillie buffer solution, APS was performed using an Alkaline Phosphatase Detection Kit (SCR004; Alkaline Phosphatase Detection Kit, Merck Millipore).
Alkaline phosphatase detection kit
Manufactured by Merck Group
Sourced in United States, Germany
The Alkaline Phosphatase Detection Kit is a laboratory tool designed to detect and quantify the presence of the enzyme alkaline phosphatase. Alkaline phosphatase is an important biomarker used in various research and diagnostic applications. The kit provides reagents and protocols to enable the measurement of alkaline phosphatase activity in a wide range of sample types.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
Paraformaldehyde (pfa), by Merck Group (4 mentions)
Gelatin, by Merck Group (3 mentions)
Axio observer a1, by Zeiss (3 mentions)
Ssea 4, by Merck Group (2 mentions)
Tra 1 60, by Merck Group (2 mentions)
Tra 1 81, by Merck Group (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Ix71 microscope, by Olympus (2 mentions)
Accutase solution, by Merck Group (2 mentions)
Fv1000, by Olympus (2 mentions)
Eclipseti microscopy, by Nikon (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Axio observer a1 inverted light microscope, by Zeiss (2 mentions)
Axioplan microscope, by Zeiss (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (1 mentions)
140 protocols using alkaline phosphatase detection kit
1
Alkaline Phosphatase Detection in Samples
2
Adipogenic and Osteogenic Differentiation of ADSCs
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ADSCs were cultured in complete culture media in 6-well culture plates. Adipogenic differentiation was induced for 7 days in DMEM/F12 (Catalogue Number: SH30023.01, Hyclone) supplemented with 10% fetal bovine serum (FBS, Catalogue Number: S601P-500, SERA-PRO, South America), 1 μm dexamethasone (Catalogue Number: D4902, Sigma-Aldrich), 10 μm insulin (Catalogue Number: I8830, Solarbio, China), 200 μm indomethacin (Catalogue Number: 1820, Sigma-Aldrich), and 0.5 mm 3-isobutyl-1-methylxanthine (Catalogue Number: 15879, Sigma-Aldrich). Oil red O staining as an indicator of intracellular lipid accumulation was performed, as described previously [17 (link)]. For quantitative assessment, the oil red O was dissolved in isopropanol (100%) and the absorbance was measured by a spectrophotometer at 510 nm. To confirm osteogenic differentiation, ADSCs were cultured with a specific induction medium consisting of 0.1 mM dexamethasone, 10 mM β-glycerophosphate (Catalogue Number: G9422, Sigma-Aldrich), and 50 μM ascorbic acid (Catalogue Number: A4544, Sigma-Aldrich). To quantify ALP enzymatic activity, cells were cultured for 7 days, then washed three times with PBS, fixed with 4% paraformaldehyde for 2 minutes, and visualized by staining with an alkaline phosphatase detection kit (Catalogue Number: AP0100, Sigma-Aldrich).
3
Alkaline Phosphatase Staining of iPSCs
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AP staining was performed using an Alkaline Phosphatase Detection Kit (Sigma-Aldrich, St. Louis, MO, USA), following the instructions of the manufacturer. Cells were stained at day 8 after the addition of 1 µg/ml Dox when iPS
colonies had developed.
colonies had developed.
4
Alkaline Phosphatase Assay for Stem Cell Identification
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H1 and IMR90-1 cell lines and the 12 cell lines (N = 3 each sample) that were chosen for characterization were also distributed onto 1 well of 4-well plate that was pre-coated with Matrigel. Cells were allowed to grow for 4–5 days before being stained with alkaline phosphatase detection kit (Sigma-Aldrich; Merck Millipore). On the day of staining, alkaline phosphatase staining solution was prepared by mixing Fast Red Violet (FRV) with Naphthol AS-BI phosphate solution and water in a 2:1:1 ratio just before starting the staining procedure. In the beginning, cells were washed with DPBS and then fixed with 4% paraformaldehyde in PBS (Sigma-Aldrich) for 2 min. After fixative solution was aspirated and cells were rinsed with DPBS, staining solution (500 μl) was added to the cells that were then incubated in the dark for 15 min at room temperature (RT) on a microtiter shaker. Sequentially, cells were washed with DPBS and examined under a light microscope for their expression of alkaline phosphatase (red stem cell colonies).
5
Alkaline Phosphatase Staining of iPSCs
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Alkaline phosphatase staining T. osimensis iPSCs were stained using an alkaline phosphatase detection kit (Sigma) according to the manufacturer’s protocol.
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6
Alkaline Phosphatase Staining of Stem Cells
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For AP staining, cells were seeded in different densities on gelatine‐coated plates. The next day, differentiation was performed for 72 h, controls were kept in 2i + LIF medium. All cells were placed back in 2i + LIF medium. After 2–4 day, colonies were stained with the Alkaline Phosphatase Detection Kit (Sigma‐Aldrich) according to the manufacturer's instructions.
7
Quantifying Active Alkaline Phosphatase
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DP cells were lysed with ultra-pure water with 0.01% sodium dodecyl sulphate (1 h, 37 °C), followed by freezing at −80 °C. Cells were then scrapped, and DNA levels quantified using the PicoGreenTM dsDNA Assay Kit (Thermo Fisher Scientific) and the amount of active-ALP was quantified using the Alkaline Phosphatase Detection Kit (Sigma-Aldrich).
8
Maintenance and Differentiation of Mouse ESCs
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E14Tg2A, TNGA 11 , Nanog‐VNP 15 , Sox1‐GFP 21 , and T‐GFP 22 mouse ES cells were maintained or differentiated as described 23 . 2i medium used N2B27 (NDiff 227, Takara, Saint‐Germain‐en‐Laye, France) with 1 μM PDO325901 (R&D, Abingdon, UK) and 3 μM Chiron (Cambridge Wellcome Trust/Medical Research Council Stem Cell Institute). Kat2a inhibition used 100 μM MB‐3 (ab141255, AbCam, Cambridge, UK) 19 , or an equal volume of dimethyl sulfoxide (DMSO). ES cell colony‐forming capacity was quantified using Alkaline Phosphatase detection kit (Sigma Aldrich, Gillingham, UK) as per manufacturer's instructions.
9
Paraformaldehyde Fixation and Alkaline Phosphatase Detection
10
Osteoblastic Differentiation of MC3T3-E1 Cells
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The MC3T3-E1 cells were seeded at a density of 1 × 104 cells/well in 24-well plates and cultured in α-MEM supplemented with 10% FBS. After incubation for 2 d, the growth medium was changed to a medium containing 2% FBS, 100 μg/mL LF, 50 μg/mL ascorbic acid, and 10 mM β-GP. After culturing for 2 weeks, ALP activity was measured using an alkaline phosphatase detection kit (Sigma-Aldrich, Inc., St. Louis, MO, USA) according to the procedure supplied by the manufacturer, and the number of ALP-positive cells was counted in four random fields per well (n = 6 in each group).
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