To measure differences in biomass between vector control and overexpression mutants, an inoculum suspension containing approx. 5 × 107 CFU/ml was added to the wells of a round-bottomed 96-well microtiter plate (TPP). Ten wells per strain were included and the experiment was performed at least three times. Biofilms were grown in LBB supplemented with Tp and 0.2% (w/v) rhamnose to induce expression of the respective small proteins. Following four hours of adhesion, the supernatant was removed and the plates were rinsed with physiological saline (0.9% w/v NaCl,PS). Subsequently, 100 μl of fresh LBB supplemented with Tp and 0.2% (w/v) rhamnose was added and the plates were further incubated at 37 °C. After 24 h, the supernatant was removed, wells were rinsed with 100 μl PS and 100 μl of a 99% (v/v) methanol solution (Sigma) was added for 15 min. Methanol was removed and plates were dried at 37 °C. When all residual methanol was evaporated, 100 μl of a 0.1% (v/v) crystal violet stain (Prolab Diagnostics) was added for 20 min. Plates were rinsed with water and 150 μl of a 33% acetic acid solution was added for five minutes. After shaking, absorption was measured at 590 nm [28 (link)].
Methanol solution
Manufactured by Merck Group
Sourced in United States
Methanol solution is a clear, colorless liquid chemical compound. It is a common solvent used in various industrial and laboratory applications. The core function of methanol solution is to act as a solvent, providing a medium for dissolving, suspending, or diluting other substances.
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Lab products found in correlation
Crystal violet stain, by Pro-Lab Diagnostics (1 mentions)
8 μm pore insert, by Merck Group (1 mentions)
Glacial acetic acid, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Tryptic soy broth (tsb), by Merck Group (1 mentions)
Crystal violet solution, by Liofilchem (1 mentions)
Ckx53 inverted microscope, by Olympus (1 mentions)
Ep50 microscope digital camera, by Olympus (1 mentions)
Superpicture polymer detection kit, by Thermo Fisher Scientific (1 mentions)
Silica gel plate, by Merck Group (1 mentions)
Millex lg, by Merck Group (1 mentions)
Multiskan go microplate spectrophotometer, by Thermo Fisher Scientific (1 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (1 mentions)
9 protocols using methanol solution
1
Quantifying Biofilm Biomass Differences
2
Transwell Migration Assay for HUVECs
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HUVECs (3 × 104/well) were resuspended in supplemented RPMI medium. Cells were placed into the top chamber of 8-μm pore insert (Millipore), while the bottom well was filled with RPMI 1640 medium containing rGal1 (1 μM), VEGF (20 ng/mL), or plasma samples obtained from patients at different time periods following bevacizumab treatment. Alternatively, plasma samples from melanoma patients were preincubated for 1 h with human anti-Gal1 mAbs (clone 21 or 42) or isotype control. After 18 to 24 h, inserts were stained with 0.1% crystal violet and 20% methanol solution (Sigma) for 10 min at room temperature. After washing and removal of nonmigrating cells from the top chamber side, inserts were photographed and analyzed in an inverted microscope. For each insert, four images were collected, and cells were counted using Fiji 2.0 software (78 (link)).
3
Tryptic Soy Broth Glucose Vitamin E Assay
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Tryptic Soy Broth (Sigma Aldrich), glucose (Sigma Aldrich), vitamin E (α-Tocopheryl acetate) (≥96%, 0.95 g/ml, Sigma Aldrich), 96 well cell culture plate flat bottom (Orange Scientific), NaCl (Sigma Aldrich), methanol solution (for HPLC, ≥99.9%, Sigma-Aldrich), glacial acetic acid (≥99.85%, Sigma Aldrich), 2% crystal violet solution (from the Gram color kit Liofilchem).
4
Serum Metabolite Extraction for LC-MS
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Approximately 100 μL of serum was placed in an Eppendorf (EP) tube followed by the addition of 400 μL of a methanol solution (Sigma-Aldrich, St. Louis, MO, USA) with an 80% volume fraction. Next, the tube was scrolled and shaken, and thereafter, placed on ice for 5 min. This was followed by centrifugation at 15,000 ×g and 4°C for 20 min, after which the supernatant was collected and added to ultrapure MS water, and the methanol content was diluted to 53%. Centrifugation was again performed at 4°C and 15,000 ×g for 20 min. Thereafter, the supernatant was collected and analyzed via liquid chromatography-mass spectrometry (LC-MS; Agilent Technologies, Santa Clara, CA, USA) using a Hypesil GOLD column (C18) at a flow rate of 0.2 mL/min and a temperature of 40°C. In the positive mode, mobile phase A was 0.1% formic acid, while mobile phase B was methanol and in the negative mode, mobile phase A was 5 mM ammonium acetate (pH 9.0), while mobile phase B was methanol. The conditional scanning range of the MS, i.e., the mass charge ratio (m/z) range, was 100–1,500. The process of metabolite identification normalization was conducted by Novogene (Beijing, China) with mzCloud, mzVault, and MassList databases as the reference libraries.
5
Quantifying Cell Adhesion After Mechanical Stress
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Right after the mechanical treatment, the stretched samples and control counterparts were trypsin-detached, pelleted, and processed (Supplementary Figure S7 ). A cell adhesion assay after mechanical stimulus was performed on a 96-well plate that had been precoated with rat tail collagen I at a 50 µg/mL concentration and maintained at 37 °C for 2 h. SAOS-2 cells were seeded in DMEM high glucose at a density of 400 cells per well and maintained at 37 °C for 24 h under conventional tissue-growing conditions. The following day, the cells were washed and cell live confluence was recorded with the live imaging program of a TECAN sparkR multimode reader (Tecan Group Ltd., Männedorf, Switzerland). Cell monolayers were then crystal-violet-stained through 10 min of incubation in 0.5% crystal violet dissolved in 20% v/v methanol solution at 37 °C (Sigma Chemical Co., St. Louis, MO, USA). After several washes to remove the excess crystal violet, pictures were taken by an Olympus CKX53 inverted microscope equipped with an EP50 Microscope Digital Camera (Olympus Life Science, Waltham, MA, USA). Cell counts were based on counts of ten fields from digital images taken randomly at ×4 magnification by the ImageJ software ImageJ bundled with 64-bit Java 8 (ImageJ bundled with 64-bit Java 8, http://rsb.info.nih.gov/ij/ , accessed on 1 February 2023).
6
Bax Activation in Saos-2 Cells
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The Saos-2 cells were incubated for 72 h under standard conditions in the control (no treatment) and the IMH, LDOX, or LDOX plus IMH groups and then fixed with a methanol solution (Sigma-Aldrich, USA) and acetone (Chimreserv, Kyiv, Ukraine). The cells were subsequently probed with primary antibodies to Bax (clone 6A7, Thermo Scientific, Waltham, MA, USA), which recognize an epitope of the activated protein form [94 (link),95 (link)], and incubated at room temperature for 1 h. An immunocytochemistry assay provided an integral visualization of the Bax expression in the cells. Immunolabeling was detected using a SuperPicture Polymer Detection Kit (Thermo Fisher Scientific, USA) with a hematoxylin counterstain for 2 min. Bax protein expression was assessed by an H-score [96 (link)].
7
TLC Analysis of Fungal Phenazine Extracts
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The ethyl acetate extracts were air dried by rotary evaporator at 50 °C. The crude extracts of FPs were analyzed by TLC. On silica gel plates (Merck, Germany), 1 mg of commercial phenazine and crude extracts from the FPs were spotted with methanol solution (Sigma Aldrich, Mumbai). The TLC plates were developed with a solvent system of hexane: ethyl acetate at 4:2 ratio (Perneel et al., 2007 (link)). The spots were detected under UV at 254 nm and the Rf values of the crude metabolites were determined by compared with that of commercial phenazine.
8
Curcumin-loaded Gemini Surfactant Nanoparticles
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Curcumin-loaded NPs were synthesized by a single-step nano-precipitation method as previously described by our group. In brief, 6 mg of Cur (Cat no: Sigma-Aldrich, USA) and 100 mg of methoxyl-poly(ethylene glycol) urethane Gemini surfactant (provided as a gift from Institute for Color Science and Technology, Tehran, Iran) were dissolved in 5 ml of methanol solution (Merck). Then, the methanol was evaporated in a rotary evaporator. The Gemini-Cur NPs solution were filtered using a 0.2 µm size syringe filter (Millex-LG, Millipore Co., USA) to exclude the impurity and sterilize the solution Gemini-Cur NPs, were lyophilized, and stored at 4 °C until use. The characterization of Gemini-Cur NPs was done according to our previous study (Karimpour et al. 2019) .
9
Antioxidant Capacity of Mangrove Leaves
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Measurement of the antioxidant capacity of mangrove leaves was carried out using the DPPH method (Oke and Hamburger 2002) , and the assays were conducted using 96 well plate microplates. The ethanol extract of Rhizophora mucronata leaves was prepared in series concentrations of 10, 20, 40, 80 µg/mL in methanol solution (Merck). Then, 160 µL of extract from each concentration series was fed into the microplate well. Furthermore, 40 µL of DPPH (Merck) 0.76 mM solution was added to each well that contained a sample. A comparison control used vitamin C with a concentration series of 4, 6, 8, 10 µg/mL. As a control sample, each series of dilutions (160 µL) was added to the microplate well before adding 40 µL of methanol. A negative control (without extract) was made by adding 160 µL methanol with 40 µL DPPH, and 200 µL methanol was blank. The microplate was incubated in a dark room at 25-28 °C for 30 minutes, and after that, the absorbance of each well was measured with the Multiskan GO Microplate Spectrophotometer (Thermo Scientific) at a wavelength of 517 nm. The IC50 determination was measured from the inhibition percentage data in units of µg/mL, and a probit analysis was used to determine the IC50 value. The
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