Cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 15 min at room temperature, washed twice using ice-cold PBS, and developed with AST Fast Red TR and α-Naphthol AS-MX Phosphate (N4875, Sigma-Aldrich, Shanghai, China) according to the instructions [35 (link),36 (link),37 (link)]. The cells were incubated with the mixture (1.0 mg/mL Fast Red TR and 0.4 mg/mL Naphthol AS-MX in 0.1 mol/L Tris-HCL Buffer) at room temperature for 20 min. The AP-positive colonies were then shown in red. The images were captured by a Nikon phase-contrast microscope.
α naphthol as mx phosphate
Manufactured by Merck Group
Sourced in United States
α-Naphthol AS-MX Phosphate is a chemical compound used in laboratory settings. It functions as a substrate for the detection of enzyme activity, specifically the enzyme alkaline phosphatase.
Automatically generated - may contain errors
11 protocols using α naphthol as mx phosphate
1
Immunocytochemical Detection of AP Colonies
2
Pluripotency Assessment of Induced Pluripotent Stem Cells
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The pluripotency of E- and CON-piPSCs was determined by AP staining solution (AST Fast Red TR and α-Naphthol AS-MX Phosphate (Sigma Aldrich)) according to the manufacturer’s instruction. In brief, the piPSCs were incubated with 4% paraformaldehyde (Sangon Biotech, Shanghai, China) for 15 min and then washed twice with PBS. Afterwards, the cells were incubated with 0.1 M Tris Buffer consisting of 1.0 mg/mL Fast Red TR and 0.4 mg/mL Naphthol AS-MX for 20 min. Then, the pluripotency of piPSCs was detected by the color intensity of colonies using a phase-contrast microscope (Nikon, Tokyo, Japan) [26 (link)].
3
Assessing Pluripotent Stem Cell ALP
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The alkaline phosphatase (AP) activity of pluripotent stem cells was determined by AST Fast Red TR and α-Naphthol AS-MX Phosphate (Sigma Aldrich, USA) according to the manufacturer’s instructions. Briefly, cells were washed twice using ice-cold PBS, fixed with 4% paraformaldehyde in PBS (pH 7.4) for 15 min at room temperature, followed by washing three times using ice-cold PBS. Cells were then incubated at room temperature in the solution containing 1.0 mg/mL Fast Red TR, 0.4 mg/mL α-Naphthol AS-MX in 0.1 M Tris buffer. After 5–10 min incubation, AP positive colonies were displayed in red.
4
Alkaline Phosphatase Staining Protocol
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AP staining was performed using AST Fast Red TR and α-Naphthol AS-MX Phosphate (Sigma-Aldrich) according to the manufacturer's instructions.
5
Alkaline Phosphatase Staining of piPSCs
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Suitable piPSCs were fixed with 4% paraformaldehyde (pH 7.4) for 15 min, then washed 2–3 times with PBS. The cells were stained with AST Fast Red TR and α-naphthol AS MX phosphate (Sigma-Aldrich, USA) according to the manufacturer’s instructions. In brief, 1.0 mg/mL AST Fast Red TR, 0.4 mg/mL α-naphthol AS MX phosphate, and 0.1 mmol/L Tris-HCL 8.8 buffers were used to prepare the AP staining solution. The cells were incubated for 20 min with AP stain, which was then replaced with PBS. The AP-positive piPSC clones showed red and images were captured using a phase contrast microscope (Nikon, Japan).
6
Alkaline Phosphatase Staining of piPSCs
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The piPSCs (cultured for 5 days) were fixed in 4% paraformaldehyde (pH 7.4) at room temperature for 15 min, with AST Fast Red TR and α-naphthol AS MX phosphate (Sigma-Aldrich, USA) then used to stain the cells according to the manufacturer’s instructions. The piPSCs were incubated in 1.0 mg/mL Fast Red TR, 0.4 mg/mL α-naphthol AS-MX, and 0.1 mmol/L Tris-HCL 8.8 buffer at room temperature for 20 min. The AP-positive piPSCs clones showed red (Zhang et al., 2017 (link)). Images were obtained using a Nikon phase difference microscope.
7
Immunofluorescence and Alkaline Phosphatase Staining
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For immunofluorescence staining, cells were fixed with 4% paraformaldehyde in PBS for 15 min and blocked by 1% BSA at room temperature for 1 h. Cells were then incubated with primary anti-EpCAM antibody (1:200, ab71916, Abcam) at 4 °C overnight. After washed three times with PBS, cells were incubated with FITC conjugated secondary anti-rabbit antibody for 1 h. Nuclei were stained with 10 ug/mL Hoechst 33342 for 2 min. Images were captured under a Nikon fluorescence microscope. The alkaline phosphatase (AP) staining was determined by AST Fast Red TR and α-Naphthol AS-MX Phosphate (Sigma Aldrich) as our previously described4 (link). Images were captured by a Pentax K-50 digital camera.
8
Measuring Alkaline Phosphatase Activity
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Stem cells show high alkaline phosphatase (AP) activity, which decreased after differentiation. In this study, AP staining was used to measure AP activity. Briefly, AST Fast Red TR (F6760, Sigma Aldrich) and α-Naphthol AS-MX Phosphate (N4875-1G, Sigma Aldrich) were used to detect AP activity. Ice-cold PBS (SH30256.01B, Hyclone Laboratories) was used to wash cells twice; after being fixed with 4% paraformaldehyde, the cells were then incubated with the mixture of Fast Red TR and Naphthol AS-MX for 10 min.
9
Immunostaining of Induced Pluripotent Stem Cells
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Cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 15 minutes at room temperature, washed twice using ice‐cold PBS and developed with AST Fast Red TR and α‐Naphthol AS‐MX Phosphate (Sigma‐Aldrich) according to the manufacturer's instructions. Then the cells were incubated with the mixture (1.0 mg/mL Fast Red TR, 0.4 mg/mL Naphthol AS‐MX in 0.1 mol/L Tris‐HCL Buffer) at room temperature. After 20 minutes, the AP‐positive iPS colonies showed in red colour. The images were documented by a Nikon phase contrast microscope.14
10
Alkaline Phosphatase Activity Staining
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Alkaline phosphatase (AP) activity was detected by using AST Fast Red TR (Sigma) and α‐Naphthol AS‐MX Phosphate (Sigma) in accordance with the manufacturer's protocol. Cells were washed with phosphate‐buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 15 minutes at room temperature. The fixed cells were washed once with PBS and incubated with the mixture at room temperature for 15 minutes.15 The cells were observed and the images were captured by using a Nikon (Tokyo, Japan) inverted microscope after staining.
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