Cells in the collagen matrix were fixed using 4% paraformaldehyde phosphate buffer solution (Wako, Japan) at room temperature for 30 min. Then, the samples were washed twice with phosphate-buffered saline (PBS) and blocked using PBS containing 3% bovine serum albumin (BSA) (Sigma, USA) at room temperature for 1 h. After removing the blocking buffer, anti-mouse PHEX (Sigma) was added to the sample and incubated at 4 °C overnight. After incubation with the primary antibody, the samples were rinsed twice with PBS. Then, the sample was permeabilized with 0.1% Triton-X (MP Biomedicals, USA) for 20 min. After rinsing twice with PBS, the samples were subjected to incubation with the secondary antibody (Anti-rabbit IgG Alexa Fluor 488, Invitrogen, USA), actin (Alexa Fluor 546 phalloidin, Invitrogen), and DAPI staining at room temperature for 1 h. Finally, the samples were rinsed twice with PBS and observed under a confocal microscope (FV3000, Olympus, Japan). Imaging data were processed using Imaris (Bitplane, UK) and ImageJ (NIH, USA).
Triton x
Manufactured by MP Biomedicals
Sourced in United States
Triton-X is a non-ionic detergent commonly used in biochemical and molecular biology applications. It is effective in solubilizing and extracting membrane proteins and other hydrophobic biomolecules. Triton-X is a versatile lab equipment that can be used in a variety of experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Lumaplate, by PerkinElmer (2 mentions)
Autogamma counter, by Hewlett-Packard (2 mentions)
Na251cro4, by PerkinElmer (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde phosphate buffer solution, by Fujifilm (1 mentions)
Alexa fluor 488 anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546 phalloidin, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Fv3000, by Olympus (1 mentions)
Cd8 t cell isolation kit, by Miltenyi Biotec (1 mentions)
C casp3, by Cell Signaling Technology (1 mentions)
Click it edu imaging kit, by Thermo Fisher Scientific (1 mentions)
Alexa 488 or 546, by Thermo Fisher Scientific (1 mentions)
Myod1, by Santa Cruz Biotechnology (1 mentions)
Bz x810 fluorescence microscope, by Keyence (1 mentions)
Mab4470, by Merck Group (1 mentions)
D1033, by Merck Group (1 mentions)
Bz x800 analyzer, by Keyence (1 mentions)
Bz x analyzer, by Keyence (1 mentions)
Bz 9000, by Keyence (1 mentions)
5 protocols using triton x
1
Immunofluorescent Staining of Cells in Collagen Matrix
2
Cytotoxicity Assay for Tumor Cells
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Splenic CD8+ T cells (4 × 106/ml) were isolated from transplanted mice 21 d after allo-BMT, purified with a CD8+ T-cell isolation kit (Miltenyi Biotec), and used as effector cells. MBL-2, P185 BCR-ABL1 Arf null, and Ik6 tumor cells were labeled by incubating 2 × 106 cells with 2 MBq of Na251CrO4 (PerkinElmer Life) for 2 h at 37 °C in a 5% CO2 atmosphere and were used as target cells. After washing, 4 × 103 labeled targets were resuspended and added to triplicate wells at varying effector-to-target ratios and then incubated for 4 h. Maximal and minimum release was determined by the addition of Triton-X (MP Biomedicals) or media alone to targets, respectively. Supernatants were transferred to a Luma plate (PerkinElmer) after 4 h and 51Cr activity was determined using an autogamma counter (Packard).
3
Immunofluorescence Characterization of Myogenesis
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Cultured cells were fixed with 4% formaldehyde for 10 min at 4 °C, subsequently permeabilized in 0.1% triton-X (MP Biomedicals, USA) for 5 min at room temperature and blocked with PBS containing 10% goat serum for 1 h at 37 °C. Incubations with primary antibodies to MyHC (1:200; eBioscience, #14-6503-82), c-casp3 (1:200; Cell Signaling Technologies, Danvers, MA, USA #9664), and MYOD1 (1:200; Santa Cruz, sc-32758) were performed overnight at 4 °C. After washing with PBS, they were incubated with a secondary antibody conjugated with Alexa-488 or -546 (1:300; Molecular Probes) for 30 min at room temperature. Following the manufacturer's instructions, we performed EdU staining with a Click-iT EdU imaging kit (Invitrogen) and stained nuclei with 4,6-diamidino-2-phenylindole (DAPI). We analyzed the stained cells using a BZ-X810 fluorescence microscope (Keyence, Osaka, Japan). We calculated the fusion index as a percentage of nuclei within MyHC-positive myotubes in nine randomly selected images from three healthy individuals.
4
Cytotoxic T cell Assay for Tumor Killing
5
Immunofluorescence Analysis of Myogenic Markers
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For immunofluorescence analysis, cells were washed with PBS, fixed in 4% paraformaldehyde for 10 min at 4 °C, and subsequently permeabilized in 0.1% triton-X (MP Biomedicals, USA) for 10 min at RT. Cells were blocked with 10% goat serum for 15 min at 37 °C. Primary antibody incubations were performed overnight at 4 °C. Cells were then washed with PBS and incubated with secondary antibodies for 30 min at RT. Plates were imaged using a fluorescent microscope (BZ-9000 or BZ-X800, KEYENCE, Osaka, Japan) and BZ-X Analyzer or BZ-X800 Analyzer (KEYENCE). The following primary antibodies were used: mouse anti-MYOD1 (1:200, Santa Cruz; sc-32758), mouse anti-myogenin (1:200, Santa Cruz; sc-12732), mouse anti-dystrophin (1:30, Leica, NCL-DYS1), mouse anti-myosin heavy chain (1:50, R&D; MAB4470), mouse anti-α-Actinin (1:1000, Sigma; A7811), mouse anti-desmin (1:800, Sigma; D1033), and mouse anti-β-dystroglycan (1:100, Leica, NCL-b-DG). Alexa Fluor 546 goat anti-mouse IgG (H + L; 1:300, Invitrogen) or anti-mouse IgG, Dylight 488 (Vector Laboratories, USA; DK-2488) were used as secondary antibodies. Nuclei were stained with Hoechst 33342 (1:10,000; Thermo Fisher Scientific; H3570).
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