7-amino-4-methyl-2-quinolone was obtained from Astatech chemicals. 1,4,7-triazacyclononane (TACN) and S(−)-propylene oxide were purchased from TCI America. 7-Amino-4-(trifluoromethyl)coumarin and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer was purchased from Alfa Aesar. Human serum albumin lyophilized was obtained from Sigma life sciences. Nitric acid at 65–70% with greater than ≥99.999% purity (trace metals basis) was obtained from BeanTown Chemical. 100 ppm Fe standard solutions were purchased from Inorganic Ventures. Bovine collagen (3 mg/mL) was obtained from Advanced Biomatrix.
Bovine collagen
Manufactured by Advanced BioMatrix
Sourced in United States
Bovine collagen is a natural, animal-derived protein obtained from the skin and connective tissues of cattle. It is a key structural component in various tissues, providing strength and support. Bovine collagen can be used in a variety of laboratory applications, serving as a substrate or scaffold for cell culture, tissue engineering, and other research purposes.
Automatically generated - may contain errors
Lab products found in correlation
100 ppm fe standard solutions, by Inorganic Ventures (2 mentions)
S propylene oxide, by Tokyo Chemical Industry (1 mentions)
1 4 7 triazacyclononane tacn, by Tokyo Chemical Industry (1 mentions)
Phosphoethanolamine, by Merck Group (1 mentions)
Epidermal growth factor (egf), by Corning (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Thle 2, by Corning (1 mentions)
Thle 2, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
L glutamine, by Corning (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Mycoalert mycoplasma detection kit, by Lonza (1 mentions)
6 protocols using bovine collagen
1
Synthesis of Fluorescent Quinolone Probe
2
Comparative Analysis of Liver Cancer Cell Lines
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All cell lines were purchased from ATCC (Manassas, VA) and grown in flasks in monolayers at 37°C with 5% CO2. Four liver cancer cell lines were used, including HepG2/C3A (C3A) (derivative of HepG2, p53 WT), Hep3B2.1–7 (Hep3B, p53 null), PLC/PRF/5 (p53 dominant negative R249S mutation) , and Snu398 (p53 null), as well as four normal cell lines, THLE-2 (immortalized normal liver cells), hFOB1.19 (immortalized normal bone cells), IMR-90 (normal fetal lung cells), and BJ (normal foreskin fibroblasts). Cells were maintained in DMEM (C3A, Hep3B2.1–7, Snu398, PLC/PRF/5, and IMR-90), RPMI 1640 (Snu398), DMEM/F12 (hFOB1.19), or BEGM (THLE-2). Media was supplemented with 1% L-glutamine (Corning), 1% penicillin-streptomycin (Gibco), and 10% FBS (Atlanta Biologicals). THLE-2 cell media was also supplemented with 5 ng/mL epidermal growth factor (Corning) and 70 ng/mL phosphoethanolamine (Sigma-Aldrich), and the gentamycin/amphotericin and epinephrine provided with the BEGM media kit (Lonza, Walkersville, MD) were discarded as per ATCC instructions. THLE-2 cells were grown in flasks coated in fibronectin (Sigma-Aldrich), bovine serum albumin (Sigma-Aldrich), and bovine collagen (Advanced Biomatrix) as per ATCC instructions. All experiments were conducted between cell passages 5 and 20.
3
Culturing Human Melanoma Spheroids
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Human wild-type MV3 melanoma cells, kindly provided by G. van Muijen (Department of Pathology, Radboud University Medical Center, Nijmegen; van Muijen et al., 1991 (link)), were cultured (humidified atmosphere, 10% CO2, 37°C) in Dulbecco's modified Eagle's serum medium (DMEM, Invitrogen) supplemented with 10% fetal calf serum (Sigma), penicillin (100 U/ml, PAA), streptomycin (100 µg/ml, Invitrogen), L-glutamine (2 mM, Lonza) and sodium pyruvate (1 mM, Gibco). Identity of the MV3 cells was verified by short tandem repeat (STR) DNA profiling (IDEXX BioResearch), and no mammalian interspecies contamination was detected. Lack of contamination with mycoplasma was routinely verified using the MycoAlert Mycoplasma Detection Kit (Lonza). Cells were detached with EDTA (2 mM, Invitrogen), and multicellular spheroids were generated according to the hanging drop method (Korff and Augustin, 1998 (link)). Briefly, hanging droplets of a 30 µl volume containing 5000 cells, methylcellulose (40% dissolved in DMEM; Sigma) and bovine collagen (10 µg/ml; Advanced Biomatrix) were incubated for 24 h to ensure multicellular aggregation. Spheroids were washed and resuspended in medium.
4
Visualizing T Cell Apoptosis Dynamics
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T cells were labeled at days 6–8 using CMAC (10 µM) cell tracker dye (Invitrogen), and glass slide chambers were constructed as previously described (Lopez et al., 2019 (link); Lopez et al., 2022 (link)). Briefly, 2 × 106 cells were mixed in 270 µl of bovine collagen (Advanced Biomatrix, Cat# 5005-100ML) at a final concentration of 1.7 mg/ml. Collagen chambers were solidified for 45 min at 37°C/5% CO2 and placed onto a custom-made heating platform attached to a temperature control apparatus (Werner Instruments). For the induction of apoptosis, 1 µM of staurosporine (Sigma, Cat# 569397-100UG) and 800 ng of TNF-a (BioLegend, Cat# 570104) in 100 µl RPMI were added on top of the solidified collagen. Cells were imaged as soon as the addition of apoptosis inducers using a multiphoton microscope with a Ti:sapphire laser (Coherent), tuned to 800 nm for optimized excitation of CMAC. Stacks of 13 optical sections (512 × 512 pixels) with 4 mm z-spacing were acquired every 15 s to provide imaging volumes of 44 mm in depth (with a total time of 60–120 min). Emitted light was detected through 460/50 nm, 525/70 nm, and 595/50 nm dichroic filters with non-descanned detectors. All images were acquired using the 20 × 1.0 N.A. Olympus objective lens (XLUMPLFLN; 2.0 mm WD).
5
Cell Culture Media Protocol
6
Nitric Acid Purity and Chemical Reagents
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Nitric acid at 65-70% with greater than ≥99.999% purity (trace metals basis) was obtained from BeanTown Chemical (Hudson, NH, USA). The 100 ppm Fe standard solutions were purchased from Inorganic Ventures (Christiansburg, Virginia, USA). Reagent grade gelatin was purchased from VWR Life Sciences (Bridgeport, NJ, USA). Nutrient broth pH 6.9 without NaCl was purchased from Millipore Sigma (Burlington, MA, USA). D-(-)-Mannitol was purchased from Alfa Aesar (Haverhill, MA, USA). Bovine collagen (3 mg/mL) was obtained from Advanced Biomatrix (Carlsbad, CA, USA). For the yeast experiments, the cultures were grown on standard YEPD [yeast extract (10 g/L), peptone (20 g/L), and dextrose (2%)] (VWR, Bridgeport, NJ, USA), in liquid or semisolid agar media. For hyphae phenotypes, SPIDER media [Nutrient Broth (20 g/L), Millipore Sigma (Burlington, MA, USA), Mannitol (20 g/L), and K 2 HPO 4 (4 g/L); pH adjusted to 7.2 using NaOH] was used.
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