For colony formation assays, cells were trypsinized into a single-cell suspension and seeded in 6-well plates at 103 per well. After 10 d of incubation, the colonies were stained with crystal violet dye. For soft agar assays, 5 × 103 cells were suspended in media containing 0.33% Select agar (Invitrogen) and plated on a bottom layer of media containing 0.5% Select agar in a 12-well plate. The cells were cultured for 2 wk before counting. All colonies were counted with a GelCount colony counter (Oxford Optronix Inc.). Images of colonies were captured at room temperature using an Advanced Microscopy Group microscope (EVOS) equipped with a 4×/0.13 NA objective lens and Micron 2.0 software (EVOS). No imaging medium was used. The images were cropped and contrast adjusted using Photoshop.
Select agar
Manufactured by Thermo Fisher Scientific
Sourced in United States, Austria
Select agar is a solidifying agent used in the preparation of culture media for the growth and isolation of microorganisms. It is derived from red seaweed and provides a stable, inert, and transparent matrix for the cultivation of various microbes.
Automatically generated - may contain errors
Lab products found in correlation
V700 photo scanner, by Epson (2 mentions)
Yeast extract, by Thermo Fisher Scientific (2 mentions)
Enzyme, by New England Biolabs (2 mentions)
Sorbitol, by Merck Group (2 mentions)
Glycerol, by Avantor (2 mentions)
D biotin, by Merck Group (2 mentions)
Zeocin, by Thermo Fisher Scientific (2 mentions)
Glucose, by Merck Group (2 mentions)
Atcc 6538, by American Type Culture Collection (1 mentions)
Atcc 25922, by American Type Culture Collection (1 mentions)
Hydroxypropyl methylcellulose, by Merck Group (1 mentions)
Luria bertani broth lb, by Thermo Fisher Scientific (1 mentions)
Milli q system, by Merck Group (1 mentions)
D glucose, by Merck Group (1 mentions)
Chitosan, by Merck Group (1 mentions)
Mz16fa, by Leica camera (1 mentions)
Gelcount colony counter, by Oxford Optronix (1 mentions)
Mc120 hd, by Leica camera (1 mentions)
Spermidine, by Merck Group (1 mentions)
Blasticidine, by InvivoGen (1 mentions)
30 protocols using select agar
1
Quantifying Cell Transformation Potential
2
Soft Agar Colony Formation
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5×104 cells were suspended in media containing 0.33% Select Agar (Invitrogen, Carlsbad, CA) and plated on a bottom layer of media containing 0.5% Select Agar. Plates were incubated at 37°C for 2-3 weeks prior to imaging. IL-6 neutralizing antibody and/or hPSC-CM, and/or mPSC-CM protein was added directly into the top agar layer during plating. Colonies were photographed and quantified using ImageJ and analysis was performed with Prism software.
3
Establishment and Characterization of Pancreatic Cancer Cell Lines
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L3.6sl and L3.6pl pancreatic cancer cells were kindly provided by Dr. J. Fidler [43 (link)]; Panc1 cells were purchased from the American Type Culture Collection (ATCC No. CRL-1469). For 2D (2-dimensional) cell culture, L3.6sl, L3.6pl, and Panc1 cells were grown in Dulbecco´s Modified Eagle Medium (DMEM) (PAA Laboratories GmbH, Pasching, Austria) supplemented with 10% fetal bovine serum (FBS) (PAA Laboratories GmbH), penicillin (62.5 µg × mL−1, Invitrogen, Carlsbad, CA, USA), and streptomycin (100 mg × mL−1, Invitrogen) at 37 °C in a humidified atmosphere containing 5.0% CO2. For 3D cell culture, L3.6sl, L3.6pl, and Panc1 cells were seeded at clonal density in 12-well plates in 800 µL 0.40% Select Agar on top of 800 µL 0.50% Select Agar (Life Technologies, Vienna, Austria), according to standard protocols, to prevent cell adherence. The agar layers were covered with 400 µL culture medium. For L3.6sl and L3.6pl, 1 × 104 cells and, for Panc1, 5 × 103 cells were seeded and grown for 6 weeks at 37 °C in a humidified atmosphere containing 5.0% CO2. For details of TIC enrichment and characterization (tumor-initiating capacity, stemness gene expression signatures, and self-renewal), see Reference [37 (link)]. Sphere growth was documented on a stereomicroscope with NIS image capture system and quantified using Colony Counter (Microtech Nition, Funabashi City, Japan).
4
Cultivation of S. aureus and E. coli
5
Bacillus subtilis Culture Conditions
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All B. subtilis strains used in this study are listed in Table 1 . For routine growth B. subtilis was cultured in liquid LB (10 g of NaCl, 5 g of yeast extract, 10 g of tryptone per liter) or on LB plates solidified with 1.5% (w/v) select agar (Invitrogen) at 37 °C. For growth in polyamine-free medium, B. subtilis was grown in liquid MSgg (5 mm potassium phosphate and 100 mm MOPS at pH 7.0 supplemented with 2 mm MgCl2, 700 μm CaCl2, 50 μm MnCl2, 50 μm FeCl3, 1 μm ZnCl2, 2 μm thiamine, 0.5% glycerol, and 0.5% glutamate) (31 (link)), or on MSgg plates solidified with 1.5% select agar. Antibiotics were used at the following concentrations as required: 100 μg/ml of spectinomycin, 5.0 μg/ml of chloramphenicol, and 10 μg/ml of kanamycin. Standard polyamines were obtained from Sigma, and sym-homospermidine was a kind gift from Patrick Woster (Medical University of South Carolina).
6
Synthesis of Antimicrobial Hydrogel Composites
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Chitosan (CS, medium molecular weight, deacetylation degree: 75–85%), hydroxypropyl methylcellulose (HPMC, hydroxylpropoxyl content: ~9%), silver nitrate (AgNO3), N,N’-methylenebisacrylamide (MBA), tetramethylethylenediamine (TMEDA), ammonium persulfate (APS), and d-glucose were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acrylamide (AAm) was obtained from Fischer Scientific (Toronto, ON, Canada). All chemicals were used without further purification. The water used in all experiments was purified using a Millipore Milli-Q system. Luria-Bertani broth (LB) and select agar were acquired from Invitrogen (Carlsbad, CA, USA).
7
Soft-agar Spreading Assay Protocol
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Spreading ability in complex environments was carried out in soft-agar medium. LB medium supplemented with 0.15–0.25% (wt vol−1) select agar (Invitrogen) was heated carefully and cooled to 30–40 °C before pouring the plates. After solidification 2 µl of exponentially grown cultures were spotted on the plates and incubated in a moist environment over night at room temperature. Plates were scanned before the swim colonies merged using an Epson V700 Photo Scanner. Cells to be directly compared were always incubated on the same plate.
8
Screening Arsenic Tolerance in Chlamydomonas
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C. reinhardtii CliP mutants (LMJ.RY0402. 109886 and LMJ.RY0402.043400) and wild-type strain CC-5325 were ordered from the Chlamydomonas Resource Center. Cultures were maintained in Tris-Acetate-Phosphate (TAP) agar plates and liquid culture under continuous light. Agar (Invitrogen Select Agar) was washed to remove impurities. Growth screens were performed by suspending actively growing cells in liquid TAP to the same cell densities and spotting equal volumes of each suspension onto TAP agar plates without or with sodium arsenate (10–320 µM). Plates were incubated at 25 °C and 50 µE m−2 s−1.
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C. reinhardtii CliP mutants (LMJ.RY0402. 109886 and LMJ.RY0402.043400) and wild-type strain CC-5325 were ordered from the Chlamydomonas Resource Center. Cultures were maintained in Tris-Acetate-Phosphate (TAP) agar plates and liquid culture under continuous light. Agar (Invitrogen Select Agar) was washed to remove impurities. Growth screens were performed by suspending actively growing cells in liquid TAP to the same cell densities and spotting equal volumes of each suspension onto TAP agar plates without or with sodium arsenate (10–320 µM). Plates were incubated at 25 °C and 50 µE m−2 s−1.
9
Bacillus subtilis Biofilm Formation Protocol
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B. subtilis strains were grown on MSgg medium (5 mM potassium phosphate and 100 mM Mops at pH 7.0 supplemented with 2 mM MgCl2, 700 μM CaCl2, 50 μM MnCl2, 1 μM ZnCl2, 2 μM thiamine, 0.5% glycerol, and 0.5% glutamate) (9 (link)) solidified with 1.5% select agar (Invitrogen) at 30 °C at the indicated time points. FeCl3 was added at the indicated concentration and where not specified was used at 50 μM. When appropriate 5-bromo-4-chloro-3-indolyl β-d -galactopyranoside (X-Gal) was added at 120 μg⋅mL−1. To set up a biofilm, a 3-mL aliquot of LB medium was inoculated with an individual colony taken from an overnight plate and grown at 37 °C to an OD600 of 1. Unless otherwise stated, 5 µL of the culture was placed onto an MSgg plate which was incubated at 30 °C for morphology and hydrophobicity studies. Images of colony biofilms were recorded using a Nikon D3200 digital camera mounted on a Kaiser RS3XA copy stand or using a Leica MZ16FA stereomicroscope.
10
Bacillus subtilis Biofilm Growth
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B. subtilis strains were initially grown in LB medium (10 g NaCl, 5 g yeast extract, and 10 g tryptone per liter). Biofilms were grown on MSgg agar (5 mM potassium phosphate and 100 mM MOPs at pH 7.0 supplemented with 2 mM MgCl2, 700 μM CaCl2, 50 μM MnCl2, 50 μM FeCl3, 1 μM ZnCl2, 2 μM thiamine, 0.5% v/v glycerol, 0.5% w/v glutamate, 1.5% w/v Select Agar (Invitrogen). When appropriate, antibiotics were used as required at the following concentrations: chloramphenicol at 5 μg ml−1, kanamycin at 25 μg ml−1, spectinomycin at 100 μg ml−1, and tetracycline at 10 μg ml−1.
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