Cells were fixed on 35 mm glass-bottom dishes (Iwaki) using 4% formaldehyde solution (Sigma) for 20 min in room temperature, permeabilized using 0.2% Triton X-100 (Amersham Biosciences) for 10 min at 4 °C, blocked with 0.1% bovine serum albumin (Sigma) for 2 hr at room temperature, and incubated with dyes and antibodies. 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) was incubated for 10 min to visualize the cell nucleus, Alexa Fluor 594 phalloidin (Invitrogen) was incubated for 20 min to visualize the actin filaments, and primary anti-vinculin antibody (H-300; Santa Cruz Biotechnology) was incubated overnight then tagged with secondary Alexa Fluor 488 Goat Anti-Rabbit IgG antibody (Invitrogen) for 2 hrs to visualize the vinculin molecules. The fixed cells were visualized using a confocal microscope (LSM510; Carl Zeiss) at 20x and 63x magnifications. The image analysis of immunostaining images were performed manually using ImageJ. Circularity was calculated in a standard fashion as fcirc = 4πA/P2, where A is the area and P is the perimeter of each cell. The aspect ratio was calculated as AR = dmax/dmin, where dmax is the largest diameter, and dmin is the smallest diameter orthogonal to dmax.
Triton x 100
Manufactured by Cytiva
Sourced in United States, United Kingdom
Triton X-100 is a non-ionic detergent commonly used in biochemical and cell biology applications. It functions as a surfactant, capable of solubilizing and disrupting cell membranes and protein complexes. Triton X-100 is often used in buffer solutions for cell lysis, protein extraction, and immunoprecipitation experiments.
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Lab products found in correlation
Lsm 510, by Zeiss (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Alexa fluor 594 phalloidin, by Thermo Fisher Scientific (2 mentions)
Formaldehyde solution, by Merck Group (2 mentions)
Alexa fluor 488 goat anti rabbit igg antibody, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Triton x 100, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
35 mm glass bottom dishes, by Iwaki (1 mentions)
Alexa fluor 488 goat anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
Primary anti vinculin antibody, by Abcam (1 mentions)
Anti non muscle myosin iia antibody, by Abcam (1 mentions)
35 mm glass bottomed dish, by Iwaki (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Thermo Fisher Scientific (1 mentions)
Methyl green, by Merck Group (1 mentions)
N propyl gallate p3130, by Merck Group (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
Las x, by Leica (1 mentions)
Tris hcl, by Cytiva (1 mentions)
Zncl2, by Merck Group (1 mentions)
10 protocols using triton x 100
1
Immunostaining and Imaging of Cell Morphology
2
Visualization of Cellular Adhesion Structures
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Cells were fixed on 35 mm glass-bottomed dish (Iwaki) using 4% formaldehyde solution (Sigma) for 20 min at room temperature, permealized using 0.2% Triton X-100 (Amersham Biosciences) for 10 min at 4 °C, blocked with 0.1% bovine serum albumin (Sigma) overnight at 4 °C, and then incubated with dyes and antibodies. 4′,6-Diamidino-2-phenylindole, dihydrochloride (DAPI; Invitrogen) was incubated for 10 min to visualize the cell nucleus, Alexa Fluor 594 phalloidin (Invitrogen) was incubated for 1 hr to visualize the actin filaments, and primary anti-vinculin antibody (Abcam) or anti-non-muscle myosin IIA antibody (Abcam) was incubated overnight then tagged with secondary Alexa Fluor 488 goat anti-mouse IgG antibody (Invitrogen) or Alexa Fluor 488 goat anti-rabbit IgG antibody (Invitrogen), respectively, for 2 hr to visualize the target molecules. The fixed cells were visualized using a confocal microscope (LSM510; Carl Zeiss) at 63× magnification. Quantitative analysis of FAs was done by using ImageJ software.
3
Immunofluorescence Staining of CFBE Cells
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Polarized CFBE cells were fixed with PFA 4% (vol/vol), permeabilized with Triton X-100 (17-1315-01; Amersham Biosciences) 0.5% (vol/vol), and blocked with BSA 1% (wt/vol) before being removed from their supports using a scalpel. Cells were then incubated overnight at 4°C with primary antibodies, after which a mix of the secondary antibodies and nuclear dye (4 μg/ml, Methyl Green, 67060; Sigma-Aldrich) was applied for 2 h at RT. Negative controls were performed for each experiment by adding BSA instead of primary antibodies (Fig S7 ). Filter sections were mounted in a mix of N-propyl gallate (P3130; Sigma-Aldrich) and Glycerol for microscopy (104095; Merck). A list of primary antibodies can be found in Table S1.
Imaging was performed with a Leica TCS SP8 confocal microscope, using HC Plan Apo 20x/0.75 and HC Plan Apo 63x/1.4 objectives. Software used for acquisition was Leica's LAS x, and image processing was performed on ImageJ FIJI (81 (link)). FIJI was used to generate average image projections.
Imaging was performed with a Leica TCS SP8 confocal microscope, using HC Plan Apo 20x/0.75 and HC Plan Apo 63x/1.4 objectives. Software used for acquisition was Leica's LAS x, and image processing was performed on ImageJ FIJI (81 (link)). FIJI was used to generate average image projections.
4
Gelatin Zymography of Cancer Cell MMP Production
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gelatin zymography was used to evaluate the capability of the cells to produce matrix metalloproteinases (MMPs) as this is essential for interaction between cancer cells and the extracellular matrix. PSU-SK-1 cells were starved by culturing in the medium without FBS for 24 hours before collection of the condition medium. The condition medium was mixed with 5X SDS sample buffer before separation in a 120 g/L SDS-PAGE containing 1 mg/mL gelatin (Sigma, St. Louis, MO, USA). After electrophoresis at 200 V for 1 hour, the gel was washed in a 25 mL/L of Triton X-100 (Amersham, Piscataway, NJ, USA) solution twice. The gel was then incubated in buffer containing 50 mmol/L Tris-HCl (Amersham, Piscataway, NJ, USA), pH 7.5, 10 mmol/L CaCl2 (Merck, Denmark), 1mmol/L ZnCl2 (Merck, Denmark), 10 mL/L Triton X-100 for 16-18 hours, after which the gel was stained with 5 g/L Coomassie blue in 300 mL/L methanol and 100 mL/Lactic acid. After destaining, the clear band of gelatinolytic activity was documented for size determination using Bio-rad GS700 gel scanner (Bio-rad, Hercules, CA, USA). Conditioned media from A569 was used as a positive control and DMEM without FBS was used as a negative control.
5
Quantifying Cell Proliferation on HA Hydrogels
6
Comprehensive Protein Quantification Workflow
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Triton X-100 and a Strata-X 33u Polymeric Reversed Phase column were separately purchased from Amersham Biosciences (Waukesha, WI, USA) and Phenomenex (Los Angeles, CA, USA). The Bicinchoninic Acid (BCA) Protein Assay Reagent kit and triethylammonium bicarbonate buffer were respectively acquired from Pierce (Thermo Fisher Scientific, Inc., Rockford, IL, USA) and Sigma-Aldrich (Supelco, Bellefonte, Pennsylvania, USA). ZipTip Pipette tips and Milli-Q water were obtained from Millipore (Billerica, MA, USA). The iTRAQ Reagent-8Plex Multiplex kit and Trypsin Gold, mass spectrometry grade were respectively acquired from Applied Biosystems (Carlsbad, CA, USA) and Promega (Madison, WI, USA). All the other reagents were obtained from commercial sources.
7
In Vitro Heme-Based Cytotoxicity Assay
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The heme used in vitro assays was commercially obtained from Frontier Scientific (Logan, UT, USA). Drug was diluted immediately before use in 0.1 N sodium hydroxide (NaOH) and RPMI 1640 medium (Gibco, Carsbad, CA, USA) and adjusted to the concentration of 30 µM. DTT, DFO, Iron (II) sulfate hydrate (FeSO4.7H2O) and hemin for parasite culture were commercially obtained from Sigma-Aldrich (New Road, Gillingham, United Kingdom). FeSO4.7H2O was diluted in distilled water and adjusted to a concentration of 100 µM, the compound in solution releases the ferrous iron ions (Fe+2). TritonX-100 was purchased from Amersham Biosciences (Piscataway, NJ, USA). Cytotoxicity Detection Kit was from Roche Applied Science (Penzberg, Germany). LDH ELISA kit was from Wuxi Douglin Sci. (Wuxi, China). H2DCFDA fluorescent probe was from Invitrogen/Molecular Probes (Grand Island, NY, USA). Superoxide dismutase-1 (SOD-1) activity kit was from Cayman Chemical Company (Ann Harbor, MI, USA). Aminoactinomycin D (7-AAD) and Annexin V-labeled Ab were purchased from BD Biosciences (San Jose, CA, USA).
8
Trained Immunity Induces DNA Damage Response
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WT and MGMT KO BMMs were cultured at 4 × 105 cells/well in 4-well chamber slides (Thermo Fisher Scientific). Trained immunity was induced as indicated in Figure 1 A. BMMs were fixed with 4% paraformaldehyde (Sigma-Aldrich, USA) after BG treatment and LPS restimulation. Samples were blocked and permeabilized with 5% normal goat serum (Cell Signaling Technology, USA) and 0.3% Triton X-100 (Amersham Biosciences, Little Chalfont, UK) in PBS buffer. The antibodies against mouse anti-phosphor-ATM (Biolegend, USA) and rabbit anti-γH2AX (Cell Signaling Technology, USA) were diluted in 1% bovine serum albumin (Sigma-Aldrich, USA), 0.3% Triton X-100 in PBS buffer at 1:500. After incubation overnight, fluorescent dye-conjugated secondary antibodies, including anti-rabbit IgG antibody conjugated with Alexa Fluor 488 and anti-mouse IgG antibody conjugated with Alexa Fluor 555 (both from Cell Signaling Technology, USA), were used for detection. Nuclei were stained with Hoechst 3342 (Thermo Fisher Scientific, USA). Images were acquired with a Confocal microscope LSM 980 with Airyscan 2 (Carl Zeiss AG, Germany) and prepared by ZEISS ZEN 3.5 (blue edition, Carl Zeiss AG, Germany). Mean fluorescence intensity (MFI) in each condition was measured from at least 90 positive cells by ImageJ analysis.
9
Quantifying Apoptosis and Cell Cycle in MDA-MB-231 Cells
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MDA-MB-231 cells were seeded in 10 cm dishes with 1 × 106 cells per dish and transfected with pCIRC2T7-circAAGAB plasmids for 24 h. Cells for examining apoptosis were harvested and stained by using a FITC Annexin V Apoptosis Detection Kit I (PharMingen, BD Biosciences, NJ, USA) according to the manufacturer’s protocols. Cells for examining the cell cycle were harvested and fixed by 75% ethanol at − 20 °C overnight, then lysed with 0.5% Triton X-100 (Amersham, Little Chalfont, Buckinghamshire, UK), subjected to RNase A (Qiagen) treatment (20 ng RNase A/mL in PBS) and stained with PI (BD Biosciences, NJ, USA) solution (20 μg PI/mL in PBS) in the dark for 10 min. Afterwards, cell cycle and cell apoptosis were detected by a Beckman Coulter FC500 instrument (Beckman Coulter, Inc.) using CXP Analysis Software v2.3 (Beckman Coulter, Inc.).
10
Gelatinase Activity in Cardiomyopathy
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Activities of MMP-2 and MMP-9 in myocardium tissue sample from CCC, DCM and Control (5 samples/each group) were examined by gelatin zymography. About 70 µg protein isolated from each heart tissue sample were applied to 1D electrophoresis using 10% polyacrylamide gel copolymerized with 1% gelatin type A from porcine skin (Sigma, USA) as a substrate for gelatinolytic proteases. After electrophoresis, the gels were washed twice in 2.5% Triton X-100 (Amersham Biosciences, UK) in water for 30 min. The gels were then incubated overnight at 37°C in reaction buffer (5 mM CaCl2, 2 mM NaN3 and 50 mM Tris–HCL buffer pH 7.4). After incubation, the gels were stained for 3 h in 45% methanol,10% glacial acetic acid containing 1% Coomassie Blue R-250 (Amersham Biosciences/GE Healthcare, USA) and were subsequently partially destained with the same solution without dye. The gelatinolytic activity of each MMP was qualitatively evaluated as a clear band of digested gelatin against a blue background. To confirm that the visualized bands of lysis were due to MMP activity, selected duplicate gels were incubated with MMP inhibitors (5 mM EDTA or 10 mM 1,10-phenanthroline). Proteolytic signals of active MMP-2 and MMP-9 were quantified based on the peak area, using the software ImageQuant TL (Hu and Beeton, 2010 (link)).
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