Two pathogenic fungi namely, C. albicans (ATCC 10231) and M. furfur (ATCC 14521) were used in this study. They were maintained and cultured according to the CLSI recommended methods. The micro dilution method was used to determine the MIC of tested samples (24 (link)). A stock solution of 1.0 g/ml for each tested sample was prepared in dimethyl sulfoxide. These stock solutions were two fold serially diluted with the RPMI 1640 medium (Life Technologies, Gibco®) into five different concentrations (50, 25, 12.5, 6.45, 3.125, 1.562 mg/ml). Subsequently, these tested samples in different concentrations were evaluated in 96-well plate for antifungal activity. Briefly, 40 μl of the tested samples were placed in each well and 10 μl of fungal suspension was added to each well. The positive control comprised of 40 μL of RPMI medium and 10 μL of fungal suspension, whereas the negative control contained 50 μL of RPMI medium. The micro plates were incubated at 37°C for 24 h (24 (link)). After that, 10 μL p-iodonitrotetrazolium violet (Sigma Aldrich, USA) (2 mg/mL, in water) was added to each well and the micro plates were further incubated at 37°C for 48 h. MIC was defined as the lowest concentration that inhibited the colour change of p-iodonitrotetrazolium violet (25 (link)).
P iodonitrotetrazolium violet
Manufactured by Merck Group
Sourced in United States, Germany, Sao Tome and Principe, Israel
P-iodonitrotetrazolium violet is a chemical compound used as a redox indicator in various laboratory applications. It is a pale yellow crystalline powder that changes color depending on the oxidation-reduction state of the system it is used in. The core function of this compound is to serve as a visual marker for such reactions.
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Lab products found in correlation
Noble agar, by Merck Group (5 mentions)
Gallic acid, by Merck Group (3 mentions)
Celltiter glo luminescent cell viability assay, by Promega (2 mentions)
Mquant spectrophotometer, by Agilent Technologies (2 mentions)
Methocult methylcellulose solution, by Merck Group (2 mentions)
Kojic acid, by Merck Group (2 mentions)
Aluminum chloride, by Merck Group (2 mentions)
Hydrochloric acid (hcl), by Merck Group (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
Potassium persulfate, by Merck Group (2 mentions)
Folin ciocalteu reagent, by Merck Group (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Mueller hinton broth (mhb), by Merck Group (1 mentions)
Cerulein, by Merck Group (1 mentions)
Rat anti mouse neutrophil, by Bio-Rad (1 mentions)
Butylated hydroxytoluene, by Merck Group (1 mentions)
Thiobarbituric acid, by Merck Group (1 mentions)
47 protocols using p iodonitrotetrazolium violet
1
Antifungal Activity Assessment of Tested Samples
2
Bioautography for Plant Extract Bioactivity
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The bioautography procedure as described by Begue and Kline [39 (link)] and refined for plant extracts by Masoko and Eloff [40 ] was used to identify bioactive chromatograms of plant extracts. The duplicate of TLC plates prepared were dried overnight under a stream of air to remove residual TLC solvents which may be harmful to bacteria. A 10 ml of overnight broth culture of test bacteria in Mueller Hinton broth (Merck) was centrifuged at 5300 x g for 20 min. The supernatant was discarded and the pellet was re-suspended in 2–4 ml of fresh broth and adjusted to make 0.5 McFarland standards which is equivalent to1.0 × 10−7 cfu/mL [41 ]. The dried chromatographic plates were sprayed with the test bacteria until they were completely wet in a Laminar flow cabinet (Labotec, SA). The plates were incubated overnight at 37 °C in a clean chamber at 100% relative humidity. After overnight incubation, plates were sprayed with a 2 mg/ml solution of INT (p-iodonitrotetrazolium violet, Sigma Chemicals). Plates were incubated and monitored for colour development at 2 h and further incubated overnight. Inhibition of growth of tested organisms was indicated by clear or yellow zones on chromatogram an indication of where reduction of INT to the coloured formazan did not take place.
3
Immunoblotting and Immunohistochemistry Protocols
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Our CLIC1‐specific polyclonal rabbit antibodies, AP823 and AP1089, have been previously described (Tulk and Edwards 1998 ; Ulmasov et al. 2007 ). Commercial antibodies were as follows: Rabbit monoclonal anti‐phospho ERM, mouse monoclonal anti‐ezrin, and HRP‐conjugated anti‐rabbit and anti‐mouse IgG antibodies were from ThermoFisher. Anti‐GAPDH and anti‐mouse F4/80 from Santa Cruz, rat anti‐mouse neutrophil (now known as LY‐6B.2 alloantigen) from ABD‐Serotec, and anti‐NADPH oxidase (Nox2/gp91phox) from Bioss. Fluorescent‐labeled secondary antibodies from Life Sciences, ABC kit for HRP immunohistochemistry from Vector Labs. Dihydroethidium and 6‐methoxy‐N‐ethylquinolinium iodide (MEQ) from ThermoFisher. Cerulein, folic acid, p‐iodonitrotetrazolium violet, butylated hydroxytoluene, thiobarbituric acid, and phorbol‐12‐myristate‐13‐acetate (PMA) from Sigma‐Aldrich.
4
Assessing Anchorage-Independent Growth of Engineered MCF-10 Cells
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MCF-10 series cells either (1) stably transduced with either GRM1 or control LacZ; (2) stably transduced with shGRM1 or control nonsilencing vector; or (3) treated with BAY, were assayed for anchorage-independent growth in a 0.4% agar solution in six well plates. Selection media or BAY-supplemented media was fed thrice a week for three weeks. After three weeks, colonies were stained with p-iodonitrotetrazolium violet (Sigma-Aldrich, St. Louis, MO) overnight, and counted on Gel Count machine (Oxford Optronix, Oxford, UK). For 3D live cell cultures of MCF10A series cells, 1.5×103 cells were seeded on 12 mm coverslips precoated with rBM (Cultrex®, R & D Systems, Minneapolis, MN) followed by the addition of a 2% rBM overlay [29] (link). The morphology of cells and degree of colony formation in the 3D rBM were assessed by visual examination of 16 contiguous fields under differential interference contract (DIC) microscopy at day 4 in culture as described [36] . All images were obtained using a Zeiss LSM-510 META confocal microscope.
5
Soft Agar Colony Formation Assay
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5x103 cells were suspended in 0.5% low melting agarose as top layer (SeaPlaque; Lonza, Rockland, ME, USA) and plated on the 0.7% solidified bottom layer agarose in six-well plates with duplicate. The plates were incubated at 37 °C in humidified incubator for 21 days and the 0.5 ml complete culture medium of top layer was refreshed every week. For visualization, colonies were stained with 200 μl of 0.1% p-iodonitrotetrazolium violet (I8377; Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in a humidified incubator overnight. Plates were photographed by AlphaImager HP system and the total number of colonies formed on each well were counted by AlphaEaseFC software.
6
Naphthol AS-TR phosphate disodium salts inhibit cell proliferation and colony formation
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Cells were seeded in 96 well plates at 2×103 cells/well with RPMI-1640 medium supplemented with 5% heat-inactivated FBS and without Anti-Anti. Cells were treated with Naphthol AS-TR phosphate disodium salts (NASTRp, Sigma-Aldrich, N6125) as 0–80 μmol/L for 96 hours. Cell proliferation was measured with MTT or CellTiter Glo luminescent cell viability assay (Promega, Madison, WI, USA). Cell viability of vehicle-treated cells was set to 100% of proliferation. For colony formation assay, cells were seeded in 6 well plates at 1×103 cells/well. Cells were treated with NASTRp as 0, 5, 10, and 20 μmol/L and were changed with fresh media containing NASTRp every other day for 10–17 days at which point 0.1% (wt/vol) crystal violet (Thermo Scientific, NJ, USA) was used to visualize colonies. Soft agar assay was performed as described previously [12 (link)]. Briefly, colonies were stained with p-iodonitrotetrazolium violet (Sigma-Aldrich) to select alive colonies and then, stained colonies were counted using Image J software (NIH, USA). A colony was defined as anything containing more than 10 cells, as indicated >50 pixels in Image J.
7
Soft Agar and In Vivo Tumorigenicity Assays
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Stably miR-31-transfected C666-1 and control cells were subjected to the soft agar assay for anchorage-independent growth in 4 mL medium supplemented with 0.35% agarose and layered on a 5 mL base of 0.7% agarose [41 (link)]. Experiments were carried out in triplicate. After 40 days, cells were stained with 0.8 mM p-iodonitrotetrazolium violet (Sigma-Aldrich). The in vivo tumorigenicity assay was performed as described previously [41 (link)]. A total of 2 × 106 C666-1 cells stably expressing miR-31 or controls were subcutaneously inoculated into the flank of female BALB/c nude mice (nu/nu) (4 mice/construct). Tumor growth was monitored and the tumors were excised at the end of the experiment. All experimental procedures were approved by the Animal Ethics Committee of the Chinese University of Hong Kong.
8
Anchorage-Independent Cell Growth Assay
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Bottom agar layer was prepared by mixing 2X complete DMEM with 1% Noble agar (BD) in a 12-well plate for a final concentration of 0.5% and allowed to solidify. For the top agarose layer for each well, 2,500 cells in 2X compete DMEM were resuspended in 0.6% low-melting agarose (IBI Scientific) and layered over the bottom agar layer. The next day, 100 μL of complete growth media containing test compounds was overlaid the upper layer to prevent desiccation. Media was refreshed twice weekly. After 21 days, colonies were stained with 0.1% p-iodonitro tetrazolium violet (Sigma-Aldrich), imaged using an EVOS microscope (Life Technologies), and counted.
9
Evaluating Cell Proliferation and Viability
10
Soft Agar Colony Formation
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Cell suspensions of 3000 cells/ml were prepared in 0.35% agar were plated on a 0.6% agar foundation in 6-well culture plates at 37°C. After culture for 14 to 21 days, cells were stained with 2 mg/ml of p-iodonitrotetrazolium violet (Sigma) and colonies were counted with a dissecting microscope.
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