Keratinocytes were seeded at a density of 1 × 105 per 35-mm dish containing complete OTC medium (Denova Sciences, Singapore) at day 0. On day 1, cultures were treated for 1 h with the initiating agent, MNNG, or solvent control (DMSO). The cells were washed, and the medium was replaced with complete medium for 24 h before exposure to different concentrations of H2O2. After 1 week, cells were harvested from individual dishes and passaged at a density of 1 × 105 per 35-mm dish in H2O2-containing medium. Concurrently, 2 × 104 harvested cells were embedded in 0.35% noble agar (Sigma Aldrich, St. Louis, MO, USA) and layered onto 0.7% noble agar-coated 35-mm dishes during each passage of the cells. These soft agar cultures were consistently replenished with fresh H2O2-containing medium every other day. After 1 month, viable transformed colonies were stained with 1 mg/ml thiazolyl blue tetrazolium in PBS and counted.
Noble agar
Manufactured by Merck Group
Sourced in United States, Germany
Noble agar is a high-quality agar medium used in microbiological applications. It is a gelling agent derived from red seaweed, providing a nutritionally inert substrate for the growth and isolation of a wide range of microorganisms. Noble agar exhibits excellent gel strength and clarity, making it suitable for a variety of laboratory procedures.
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Lab products found in correlation
P iodonitrotetrazolium violet, by Merck Group (5 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (3 mentions)
Thp 1, by American Type Culture Collection (3 mentions)
Hl 60, by American Type Culture Collection (3 mentions)
Sp8 laser scanning confocal microscope, by Leica (2 mentions)
Hemin, by Merck Group (2 mentions)
G csf, by GoldBio (2 mentions)
Methocult classic, by STEMCELL (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Chloramphenicol, by Merck Group (2 mentions)
Trypan blue, by Merck Group (2 mentions)
Tc20 automated cell counter, by Bio-Rad (2 mentions)
Myd88, by Cell Signaling Technology (2 mentions)
Mtt reagent, by Promega (2 mentions)
Anti il 8 antibody, by Abcam (2 mentions)
α tubulin, by Cell Signaling Technology (2 mentions)
Myd88 knockdown constructs, by Merck Group (2 mentions)
Pd08959, by Selleck Chemicals (2 mentions)
Cd11b and cd14 antibodies, by Thermo Fisher Scientific (2 mentions)
Plvx ef1alpha ires mcherry vector, by Takara Bio (2 mentions)
95 protocols using noble agar
1
Transformation Assay of Keratinocytes
2
Soft Agar Colony Formation Assay
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PC‐3 cells were first treated with either 50 nm of nontargeting control siRNA or siRNA targeting the LINC00261 or FOXA2 transcripts using the RNAiMAX reagent as described above. Twenty‐four hours after siRNA transfections, 5000 or 10 000 viable PC‐3 cells were layered in 1 mL of 0.3% Noble agar (Sigma‐Aldrich; CAS Number 9002‐18‐0) over an already solidified layer of 0.6% Noble agar per well of a six‐well plate, with 200 μL of complete media on the top to prevent desiccation. The cells were then allowed to form colonies for 14 days with media being replenished every 3 days, after which they were stained with iodonitrotetrazolium chloride (Sigma‐Aldrich; CAS Number 146‐68‐9) and imaged using a scanner. Distinct individual colonies were manually counted from three randomly chosen 10× fields per well and averaged. This assay was carried out at least two times with two replicates per condition per experiment.
3
Cell Growth and Colony Formation
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Growth curves were performed by plating 1-2 x105 cells into triplicate P60 dishes and passaging and counting cells at regular intervals. Population doublings (PDs) were calculated using the following formula PD = log (final cell number/initial cell number)/log2, and cumulative population doublings plotted. For anchorage independent growth assays 1-3 x103 cells were suspended in culture media containing 0.33% Noble Agar (Sigma Aldrich), plated on top of a base layer of culture media containing 0.6% Noble Agar. After 4 weeks colonies were stained by overnight incubation at 37°C with 1% p-iodonitrotetrazolium violet (Sigma) dissolved in 100% methanol (VWR). Colonies were visualised and counted by phase microscopy.
4
Melanoma Cell Proliferation Assays
5
Lentiviral shRNA Infection and Cell Assays
6
Soft Agar Colony Formation Assay
7
Melanoma Cell Proliferation Assays
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For vital counts, cells were stained with Trypan Blue (Sigma-Aldrich) and counted with TC20 automated cell counter (Biorad).
For colony assays, 1 × 103 Mel-ST or SK-MEL-28 cells were plated in six-well plates and cultured for 1 week. The cells were then fixed and stained for 15 min with a 1% crystal violet in 35% methanol solution. Chloramphenicol (Sigma-Aldrich) was used at a 100-200-400 µg mL-1 concentration.
For soft agar assay, 1 × 104 cells were embedded in 1 mL of RPMI 1640 containing 10% FBS and 0.35% noble agar (Sigma-Aldrich) on a base layer made of RPMI 1640 containing 10% FBS and 2% noble agar in a well of a 6-well plate. After 2 to 3 weeks of incubation, cells were fixed and stained with a crystal violet solution (0.05% crystal violet, 0.05% methanol and 0.37% PFA) for 2 h.
For colony assays, 1 × 103 Mel-ST or SK-MEL-28 cells were plated in six-well plates and cultured for 1 week. The cells were then fixed and stained for 15 min with a 1% crystal violet in 35% methanol solution. Chloramphenicol (Sigma-Aldrich) was used at a 100-200-400 µg mL-1 concentration.
For soft agar assay, 1 × 104 cells were embedded in 1 mL of RPMI 1640 containing 10% FBS and 0.35% noble agar (Sigma-Aldrich) on a base layer made of RPMI 1640 containing 10% FBS and 2% noble agar in a well of a 6-well plate. After 2 to 3 weeks of incubation, cells were fixed and stained with a crystal violet solution (0.05% crystal violet, 0.05% methanol and 0.37% PFA) for 2 h.
8
Reverse Elastin Agar Plate Protocol
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Protocol was adapted from Rust et al. [28 (link)]. Reverse elastin agar plates were comprised of two layers. The base was comprised of 0.8% (wt/vol) nutrient broth (Difco, Sparks, MD) and 2% (wt/vol) noble agar (Sigma-Aldrich, Oakville, ON) dissolved in distilled water with the pH adjusted to 7.5. The top layer contained 0.8% (wt/vol) nutrient broth (Difco, Sparks, MD), 2% (wt/vol) noble agar (Sigma-Aldrich, Oakville, ON), and 0.5% (wt/vol) elastin from bovine neck ligament (Sigma-Aldrich, Oakville, ON) dissolved in distilled water with the pH adjusted to 7.5.
9
Antimicrobial Evaluation of (NH4)2Mg(H2P2O7)2•2H2O
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The antimicrobial properties of the (NH4)2Mg(H2P2O7)2•2H2O compound were evaluated using staphylococcus aureus as a model of Gram-positive bacteria and by using two other Gram-negative bacteria, namely Escherichia Coli and Klebsiella Pneumonia. For inoculum preparation, a single bacterial colony was picked from nutrient agar using disposable sterile loop, transferred into 10 mL of nutrient broth and placed overnight in incubator shaker at 37 °C with shaking speed of 100 rpm. The bacterial cell density was measured at an optical density (OD) of 600 nm using a spectro-photometer, each inoculum prepared would contains approximately 107 cfu/ml of bacteria. Bacterial sensitivity to (NH4)2Mg(H2P2O7)2•2H2O compound is performed by employing agar well diffusion method. Three-millimeter diameter holes were made in the agar plates using 50 ml disposable pipette and different concentration varying from 5 to 20 mg of this material was placed carefully in the holes, Ampicillin (10 µg/ml) was used as a standard antibiotic. The plates were overlaid with a mixture of each bacteria with 2 ml of molten 1.5% (w./vol.) noble agar (Sigma-Aldrich) at proximately 65 °C. Finally, the plates were incubated at 37 °C for 24 h and the average diameter of the inhibition zone surrounding the holes was examined.
10
3D Cell Culture Assay for Colony Formation
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Cells were grown in 2D culture for 3 days with or without doxycycline. They were trypsinized, washed with 1X clear HBSS three times and then counted. Cells with equal seeding densities were mixed with 0.3% Noble Agar (Sigma A5431) in phenol-red-free-DMEM media containing 10% charcoal stripped FBS (Gemini), antibiotics, 10nM estrogen and with or without doxycycline. They were seeded onto 35mm dishes coated with 0.5% Nobel agar in the same media. They were then toped with the same media. Estrogen-treated growth medium with or without doxycycline was replaced every 3 days. Colonies were counted after 3-3.5 weeks.
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