PCa cross-sections were fixed in 4% paraformaldehyde (PFA), blocked with blocking buffer (Pax7: 10% goat serum, 0.03% Triton X-100 (Dow Chemical Company, Midland, MI); myogenin: 5% goat serum, 0.3% Triton X-100, 1% BSA), and then incubated overnight with rabbit anti-laminin primary antibody (Supplemental Table 1 ). Slides were then incubated with filtered secondary antibody (Supplemental Table 2 ) followed by 4% PFA. Afterward, tissues were incubated in antigen retrieval solution in a heated water bath, cooled, and then incubated in 1× PBS. Slides were blocked in blocking buffer then incubated overnight with either mouse anti-Pax7 antibody to identify quiescent and activated MuSCs or mouse anti-myogenin antibody to identify differentiating MuSCs (Supplemental Table 1 ). The following day, slides were incubated with filtered secondary antibody (Supplemental Table 2 ) followed by DAPI, and cover-slipped. Slides were washed with 1× PBS between each step, except after blocking. Stained muscle cross-sections were imaged, and cell density was calculated as described above.
Triton x 100
Manufactured by Dow
Sourced in United States
Triton X-100 is a non-ionic surfactant widely used in various laboratory applications. It functions as a detergent, emulsifier, and solubilizing agent. Triton X-100 helps to solubilize and stabilize proteins, enzymes, and other biomolecules in aqueous solutions.
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Lab products found in correlation
Fibronectin, by Dow (1 mentions)
Anti cd4 antibody, by BioLegend (1 mentions)
Anti cd3 antibody, by BioLegend (1 mentions)
Ldh cytox assay kit, by BioLegend (1 mentions)
Wst 1 assay kit, by Takara Bio (1 mentions)
Rpmi 1640, by Biowest (1 mentions)
Tienam, by Merck Group (1 mentions)
Fluorescence microscope, by Keyence (1 mentions)
Anhydrous sodium carbonate, by Thermo Fisher Scientific (1 mentions)
Sodium hydroxide (naoh), by Thermo Fisher Scientific (1 mentions)
Sodium bicarbonate, by Thermo Fisher Scientific (1 mentions)
Acetone, by Thermo Fisher Scientific (1 mentions)
Acetic acid ch3cooh, by Merck Group (1 mentions)
Hoechst 33342, by Beyotime (1 mentions)
Edu solution, by Beyotime (1 mentions)
Tcs sp5, by Leica (1 mentions)
Genios microplate reader, by Tecan (1 mentions)
Propidium iodide, by Vector Laboratories (1 mentions)
Magna lyser tube, by Roche (1 mentions)
Magna lyser, by Roche (1 mentions)
14 protocols using triton x 100
1
Immunohistochemical Analysis of Muscle Stem Cells
2
Culturing Endothelial Cells on Sylgard 184
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Monomer and Cross-linking agent (Sylgard 184®), high-glucose Modified Eagle’s Medium, Fetal Bovine Serum, Fibronectin, Triton X-100, Alexa Fluor 488® phalloidin, 4’-6-Diamidino-2-phenylindole (DAPI) and LIVE/DEAD® viability/cytotoxicity kit were obtained from Dow Corning (Midland, Michigan, USA), Gibco (New York, NY, USA), Gibco (New York, NY, USA), Sigma (St. Louis, Missouri, USA), Merck (Darmstadt, Germany), Invitrogen (Waltham, Massachusetts, USA), Invitrogen (Waltham, Massachusetts, USA) and Invitrogen (Waltham, Massachusetts, USA) respectively. Human umbilical vein endothelial cells (HUVECs) were provided from National Cell Bank of Iran, Pasteur Institute of Iran.
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Monomer and Cross-linking agent (Sylgard 184®), high-glucose Modified Eagle’s Medium, Fetal Bovine Serum, Fibronectin, Triton X-100, Alexa Fluor 488® phalloidin, 4’-6-Diamidino-2-phenylindole (DAPI) and LIVE/DEAD® viability/cytotoxicity kit were obtained from Dow Corning (Midland, Michigan, USA), Gibco (New York, NY, USA), Gibco (New York, NY, USA), Sigma (St. Louis, Missouri, USA), Merck (Darmstadt, Germany), Invitrogen (Waltham, Massachusetts, USA), Invitrogen (Waltham, Massachusetts, USA) and Invitrogen (Waltham, Massachusetts, USA) respectively. Human umbilical vein endothelial cells (HUVECs) were provided from National Cell Bank of Iran, Pasteur Institute of Iran.
3
Cucurbit[n]urils as Potential Therapeutics
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Cucurbit[n]urils (n = 6, 7 and 8) were synthesized, purified and kindly provided by the Nikolaev Institute of Inorganic Chemistry (Novosibirsk, Russia). Before the experiments, CB[n] was diluted in the culture medium RPMI-1640. CB[6] and CB[7] were prepared at a concentration 0.3, 0.5 and 1 mM; CB[8] at 0.01 mM and in a PBS solution; CB[6] and CB[7] at 0.2, 0.5, 1 and 2 mM; CB[8] at 0.01 mM. RPMI-1640 (Biowest LLC, Riverside, MO, USA), gentamicin (Dalfarma, Khabarovsk, Russia), tienam (Merck Sharp & Dohme Corp., Kenilworth, NJ, USA), FCS (Hyclone, Chicago, IL, USA), tabs for preparing phosphate-buffered saline (PBS) (AppliChem GmbH, Darmstadt, Germany), Na2EDTA (Helicon, Moscow, Russia), Ficoll (BioClot, Aidenbach, Germany), urografin 76% (Schering, Berlin, Germany), human albumin solution (Microgen, Moscow, Russia), Triton™ X-100 (Dow Corning, Midland, MI, USA, WST-1 assay kit (Takara Bio, Kusatsu, Japan), LDH-Cytox™ Assay Kit (BioLegend, San Diego, CA, USA), PE Annexin V Apoptosis Detection Kit with 7ADD (BioLegend, San Diego, CA, USA), anti-CD3 antibody (BioLegend, San Diego, CA, USA) and anti-CD4 antibody (BioLegend, San Diego, CA, USA).
The Ethical Committee of RIFCI, Russia, approved the study design and the recruitment of subjects. Subjects provided written informed consent. The relevant guidelines and regulations were followed when performing the experiments.
The Ethical Committee of RIFCI, Russia, approved the study design and the recruitment of subjects. Subjects provided written informed consent. The relevant guidelines and regulations were followed when performing the experiments.
4
Parathyroid Organoid Immunofluorescence Imaging
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Parathyroid organoids were plated in 8-well chambered slides using 75% Matrigel gel and maintained in complete organoid culture overnight. Media was aspirated, and organoids were washed and fixed with methanol (100%) and permeabilized with Triton X-100 (0.5%; Dow Chemical Company, Midland, MI) in phosphate-buffered saline (PBS). The organoids were blocked with 5% bovine serum albumin (BSA) in PBS and incubated with calcium-sensing receptor (CaSR) antibody (Sigma-Aldrich, Corp) overnight. The organoids were washed and incubated with a secondary Alexa-conjugated antibody (1:250) antibody (Invitrogen/ThermoFisher). Fluorescent images were captured using fluorescent filters with a fluorescence microscope (Keyence, Itasca, IL) with 3D analytic capabilities and a broad wavelength range (ultraviolet [UV] to infrared [IR]).
5
Quantifying Drug Loading and Encapsulation in Liposomal Vesicles
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For this study, the liposomal vesicles were disrupted by addition of methanol and Triton™ X-100 (The Dow Chemical Company, Mitland, MI, USA), and the drug contained within the vesicles was diluted in PBS for quantification by HPLC for VAN and CFZ, respectively. The concentration of drug was intrapolated on VAN and CFZ standard curves and the results were obtained in triplicates. % Drug loading and % encapsulation efficacy were calculated by the following formulae:
6
Synthesis of Organoselenium Compounds
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Acetic acid (CH3COOH, 99.7%) was obtained from Sigma Aldrich (St. Louis, MO, USA). Acetone (C3H6O, 99.5%), sodium bicarbonate (NaHCO3, 99.7%), sodium hydroxide (NaOH, 98%), and anhydrous sodium carbonate (Na2CO3, 99.5%) were obtained from Fisher Scientific (Hampton, NH, USA). A cationizing agent, 3-chloro-2-hydroxypropyltrimethyl ammonium chloride (CHPTAC), also called CR-2000 (C6H15Cl2NO, 65%), and Triton X-100 (2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol) (C16H26O2, 99+%) were obtained from Dow Chemical (Midland, MI, USA). Sodium sulfate anhydrous (Na2SO4, 99.5%) was received from Cooper Natural Resources (Fort Worth, TX, USA). The organoselenium compounds, OS-1 (C6Cl4N6Se2, >99%) and OS-2 (C6H2Cl2N6Se4, >99%), were obtained from Attach Chem (Lubbock, TX USA) and used as received. Deionized (DI) water was obtained from AquaOne (Amarillo, TX, USA) and was used for all purposes.
7
Cell Proliferation Analysis via EdU Assay
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Cells from six‐well plates were digested with trypsin and seeded into 96‐well plates at a density of 4 × 104 cells per well after 48 h of transfection. EdU solution (Beyotime, China) was diluted at a ratio of 1000:1 to prepare EdU culture medium, which was added to each well (100 μL per well). After 2 h of incubation, excess EdU medium was removed, and cells were washed with PBS. Cells were fixed with 4% paraformaldehyde, permeabilized with Triton X‐100 (Dow Chemical Company, USA), stained with Hoechst 33342 (Beyotime, China), and observed under a fluorescence microscope after incubation.
8
Cellular Uptake of Lipid Nanoparticles
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The C6 and bEnd.3 cell lines were seeded into 24-well plates at a density of 2×105 cells/ml. After 24 h, each well was subsequently incubated with 1 ml of a 100 mg/ml coumarin-6-loaded LP, RGD-LP, TF-LP and RGD/TF-LP for 2 h. For quantitative analysis, at the designated time period, the suspension was removed and the wells were washed three times with 1,000 μl cold PBS. Following this, 50 μl of 0.5% Triton X-100 (Dow Chemical Co., Midland, MI, USA) was introduced into each well for cell lysis. The fluorescence intensity of each sample well was measured using the GENios microplate reader (Tecan, Männedorf, Switzerland), with an excitation wavelength of 465 nm and emission wavelength of 502 nm. For the qualitative study, the cells were washed three times with cold PBS and fixed with 4% paraformaldehyde for 20 min. Then, the cells were washed twice with cold PBS and observed by confocal laser scanning microscopy (Leica TCS SP5; Leica, Mannheim, Germany).
9
Trace Metal Analysis of Hair and Nails
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Within the laboratory, human hair and nail samples were first thoroughly washed with 0.1% Triton-X-100 (Dow Chemical Midland, MI), and then with reagent grade water in order to remove external contaminants. The samples were then allowed to completely dry in a 90°C oven. Dried hair and nail samples were then weighed and digested using 0.5 mL of trace metal grade 65% nitric acid (Fisher Scientific, Waltham, MA) on a dry bath incubator set to 80°C. The target sample weight of 0.05–0.10 g of hair or nails was used for sample digestion. After complete digestion, 0.5 mL of a 1% HNO3 solution was added to make a final volume of 1.0 mL. Finally, prior to analysis, an internal standard solution containing gallium was added to the sample.
10
Indirect Immunofluorescence Staining Protocol
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Immunofluorescence analysis of all primary antibodies was performed via the indirect immunofluorescent technique. Sectioned samples were immediately washed with phosphate-buffered saline (PBS) and embedded in optimal cutting temperature (OCT) compound. Frozen sections were sliced to the thickness of 8 µm, placed on gelatin-coated slides, air dried, and fixed in 100% acetone or 4% paraformaldehyde at 4°C. After several washings with PBS, the sections were incubated with 1% bovine serum albumin at room temperature (RT) for 30 minutes to block nonspecific binding. They were then incubated with the appropriate primary antibodies for 1 hour at RT, and washed 3 times in PBS containing 0.15% TRITON X-100 (The Dow Chemical Company, Midland, MI, USA) for 15 minutes per wash. For negative control experiments, the equivalent serum was used. After staining with the primary antibodies, the sections were incubated at RT for 1 hour with appropriate fluorescein-conjugated secondary antibodies. After several washes with PBS, they were coverslipped with mounting medium containing propidium iodide (PI; Vector Laboratories, Inc., Burlingame, CA, USA). The slides were then examined by confocal microscopy (FluoView; Olympus Corporation, Tokyo, Japan).
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