B16BL6 murine melanoma cells (KCLB No. 8006; Korean Cell Line Bank [KCLB], Seoul, Korea) were cultured in minimum essential media alpha (α-MEM; Gibco, Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Rocky Mountain Biologicals, Missoula, MT, USA), 1% (v/v) penicillin and streptomycin (Lonza, Basel, Switzerland) on culture dishes. A431 human squamous carcinoma cells (CRL-1555; American Type Culture Collection (ATCC), MD, Manassas, VA, USA) and MDA-MB-231 human breast carcinoma cells (HTB-26; ATCC, MD, Manassas, VA, USA) were cultured in Dulbecco’s modified eagle medium (DMEM; Lonza) supplemented with 10% (v/v) fetal bovine serum (Rocky Mountain Biologicals, Missoula, MT, USA), 1% (v/v) penicillin and streptomycin (Lonza) on culture dishes. Primary human umbilical vein endothelial cells (HUVECs; CC-2517; Lonza) were cultured in endothelial cell growth medium (EGM-2; Lonza) supplemented with 10% (v/v) fetal bovine serum (Rocky Mountain Biologicals, Missoula, MT, USA) and 1% (v/v) penicillin and streptomycin (Lonza) on culture dishes. All cells were incubated at 37 °C under 5% CO2.
Penicillin and streptomycin
Manufactured by Lonza
Sourced in United States, Switzerland, United Kingdom, Germany, Belgium, France, Italy
Penicillin and streptomycin are antibiotics commonly used in laboratory settings. They function as antimicrobial agents, inhibiting the growth and reproduction of certain bacteria. This lab equipment is utilized to maintain sterile conditions and prevent contamination during various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (19 mentions)
L glutamine, by Lonza (9 mentions)
Fetal bovine serum (fbs), by Lonza (7 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (7 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (4 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (4 mentions)
Oligo dt, by Bioneer (4 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (3 mentions)
Hepes, by Lonza (3 mentions)
Rpmi medium, by Lonza (3 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (3 mentions)
Phorbol myristate acetate (pma), by Merck Group (3 mentions)
Ionomycin, by Merck Group (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 medium, by Lonza (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Welgene (3 mentions)
Dulbecco s modified eagle s medium dmem, by Welgene (3 mentions)
α mem, by Thermo Fisher Scientific (2 mentions)
70 protocols using penicillin and streptomycin
1
Culturing Diverse Cancer and Endothelial Cells
2
Culturing BSR-T7/5 and KC Cells
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BSR-T7/5 cells are a clone of baby hamster kidney-21 cells that constitutively express T7 polymerase, and were used with permission from Ulla Buchholz, Department of Clinical Virology, Federal Research Centre for Virus Diseases of Animals, Tubingen, Germany [65 (link)]. The cells were cultured in Eagle’s minimum essential medium supplemented with Earle’s Balanced Salt Solution; 2 mM L-Glutamine (HyClone™, Logan, UT, USA), 1% non-essential amino acids (Sigma-Aldrich®, Merck, Darmstadt, Germany), 5% foetal bovine serum (FBS, Gibco®, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin and streptomycin (Lonza, Basel, Switzerland). Geneticin (Thermo Fisher Scientific, Waltham, MA, USA) was added every third passage at 1 mg/mL. Cells were grown at 37 °C in the presence of 5% CO2. KC cells (derived from Culicoides variipennis) were obtained from the Onderstepoort Veterinary Institute (Pretoria, South Africa) and cultured at 28 °C in TC-100 insect medium (Lonza, Basel, Switzerland) with nonessential amino acids supplemented with 10% FBS (Gibco®, Thermo Fisher Scientific, Waltham, MA, USA), 1% penicillin and streptomycin (Lonza, Basel, Switzerland), and antifungals (Fungizone, Sigma-Aldrich®, Merck, Darmstadt, Germany).
3
Canine and Human Prostate Cancer Cell Lines
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The canine prostate cancer cell PC1 was established from a fresh tissue from a 10-year-old mixed breed dog with non-metastatic tumor and the PC2 was established from an 11-year-old poodle dog with metastatic tumor, both were previously established and characterized [44 ] in accordance with the institutional ethics guidelines. PC1 and PC2 cells at passage 20 were cultured in DMEN/F12 medium (Lonza, Basel, Switzerland) supplemented with 10% of fetal bovine serum (Lonza, Basel, Switzerland) and 1% of penicillin and streptomycin (Lonza, Basel, Switzerland). The human cell lines LNCaP (passage 15) and PC3 (passage 23) were retrieved from the European Collection of Authenticated Cell Culture (ECACC) and American Type Culture Collection (ATCC), respectively. These human cell lines were cultured in RPMI medium (Sigma Aldrich, Saint Louis, MO, USA) supplemented with 10% of fetal bovine serum (Lonza, Basel, Switzerland) and 1% of penicillin and streptomycin (Lonza, Basel, Switzerland). All cell lines were negative for mycoplasma contamination. Table 1 summarizes the main characteristics of the cell lines used in our study.
4
Vitamin D Analogs in A549 Cells
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Alveolar epithelial cells type II (A549, ATCC) were maintained in DMEM medium supplemented with 10% FBS (Sigma-Aldrich, Burlington, MA, USA), 2 mM glutamine and 100 U/mL of penicillin and streptomycin (Lonza, Basel, Switzerland). We used the active form of vitamin D (1α,25-Dihydroxyvitamin D3 or calcitriol) (cat.#D1530; Sigma-Aldrich; Vitamin D stock was 10 μM in ethanol) and the following vitamin D analogs (stocks were 50 μM in ethanol): calcipotriol (cat.#203537; Santa Cruz Biotechnology, Dallas, TX, USA), paricalcitol (cat.#477938; Santa Cruz Biotechnology), tacalcitol (cat.#sc-361371a; Santa Cruz Biotechnology, Dallas, TX, USA), 22-Oxacalcitriol (cat.#sc-361076; Santa Cruz Biotechnology, Dallas, TX, USA) and vitamin D2 (cat.#sc- sc-205988; Santa Cruz Biotechnology, Dallas, TX, USA). Treatments were performed in cells maintained in DMEM supplemented with 10% hormone-depleted serum. This serum was prepared by using the anion exchange resin AGR1-X8 from BIO-RAD (cat.#1401441; BIO-RAD, Hercules, CA, USA) as previously described [20 (link)]. Bleomycin sulfate (cat.# CAYM13877–50) was purchased to VWR [Radnor, PA, USA] (bleomycin stock: 50 mM in PBS).
5
Cell Culture and Lysis Methods
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U2OS, HeLa, mouse embryonic fibroblasts (MEFs) and HEK293 cells were maintained in a complete medium of DMEM (Gibco) supplemented with 10% foetal bovine serum (FBS, Hyclone), 2 mM L-glutamine (Lonza) and 1% penicillin and streptomycin (Lonza). Cells were cultured within a humidified environment at 37°C with 5% CO2. Wild-type and knockout RICTOR MEFs (Shiota et al, 2006 (link)) were provided by Prof. M. Magnuson (Vanderbilt University School of Medicine, USA).
For lysis, cells were washed twice on ice with PBS and subsequently scraped with ice-cold NP-40 lysis buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 1 mM EDTA, 10% (v/v) glycerol, 0.5% NP-40) unless otherwise stated. All lysis buffers were supplemented with 1 mM DTT, 1 mM PMSF and phosphatase inhibitors. Samples were clarified by centrifugation at 20,000 g for 10 min at 4°C and protein concentration determined by Bradford (500-0006, Bio-Rad protein assay).
For lysis, cells were washed twice on ice with PBS and subsequently scraped with ice-cold NP-40 lysis buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 1 mM EDTA, 10% (v/v) glycerol, 0.5% NP-40) unless otherwise stated. All lysis buffers were supplemented with 1 mM DTT, 1 mM PMSF and phosphatase inhibitors. Samples were clarified by centrifugation at 20,000 g for 10 min at 4°C and protein concentration determined by Bradford (500-0006, Bio-Rad protein assay).
6
Mesenchymal Stem Cell Culture and Stimulation
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Human bone marrow mesenchymal stem cells (MSCs; Cefobio, Korea) were cultured in DMEM growth medium containing low glucose (Gibco; USA), supplemented with 10% fetal bovine serum (FBS; Gibco), 1% penicillin and streptomycin (Lonza), and incubated at 37 °C in humidified atmosphere containing 5% CO2. Cells were harvested using 0.25% trypsin-EDTA (Gibco). Harvested cells were then seeded on a 100 mm cell culture dish with flash conditioned media. Cells were counted and 3 × 104 cells were seeded on each 100 mm dish. Seventh passage MSCs (CEFO, South Korea) were used in this study.
1 × 104 MSCs were seeded over acellular dermal matrix (ADM; L&C Seongnam-si, Korea) (1 cm × 1 cm) in a 24-well plate, and further cultured for 24 h in the above-mentioned optimal conditions. MSCs were then treated with 100 ng/mL recombinant human EGF (Gibco) or 100 ng/mL recombinant human basic FGF (Gibco) for 24 h, washed twice with PBS, and used for further experiments.
1 × 104 MSCs were seeded over acellular dermal matrix (ADM; L&C Seongnam-si, Korea) (1 cm × 1 cm) in a 24-well plate, and further cultured for 24 h in the above-mentioned optimal conditions. MSCs were then treated with 100 ng/mL recombinant human EGF (Gibco) or 100 ng/mL recombinant human basic FGF (Gibco) for 24 h, washed twice with PBS, and used for further experiments.
7
Expansion of HUVEC and ASF-2 Cell Lines
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HUVEC cells were grown in EGM Bulletkit medium (cc-3124, Lonza) in tissue culture flasks coated with 0.2% gelatin. ASF-2 cells were grown in DMEM (Lonza) with 10% fetal bovine serum (Thermo Scientific), 100 U/mL penicillin and streptomycin (Lonza) at 37 °C, 5% CO2 and 95% humidity. Cells were detached from the tissue culture flasks by trypsination with Trypsin EDTA (Lonza). Cell were passaged 1:4 at approximately 80% confluence.
8
Orthotopic Implantation of TNBC Cells in Rats
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As previously described [38 (link)], triple-negative breast cancer MDA-MB-231 cells were maintained in DMEM media (Sigma, Burlington, MA, USA) supplemented with 10% FBS (Gibco, New York, NY, USA) and 1% penicillin and streptomycin (Lonza, Cohasset, MN, USA) and incubated in 5% CO2 at 37 °C. These cells (6 × 106) in 50% Matrigel were orthotopically implanted into the mammary fat pads (MFP) of 4- to 6-week-old female Dll4+ (n = 8) and Dll4– (n = 17) rats and eight congenic strains CG1 (n = 19), CG2 (n = 2), CG3 (n = 26), CG4 (n = 12), CG5 (n = 28), CG6 (n = 12), CG7 (n = 5), and CG8 (n = 4) (Figure 1 c) [54 (link)]. Tumors were treated after 10 days of implantation at an approximate size of 600 mm3, consistent across all rat strains.
9
Phospho-Raptor Activation in HeLa Cells
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HeLa cells were obtained from the American
Type Culture Collection. The cells were cultured in Dulbecco’s
Modified Eagle Medium (DMEM; Lonza) in a humidified incubator at 37
°C with 5% CO2. To active the phosphorylation of Raptor,
the cells were cultured in either glucose-free DMEM for 24 h or in
normal DMEM with 1 mM AICAR for 1 h before the experiment. All the
DMEM was supplied with 10% fetal bovine serum (Invitrogen) and 1%
penicillin and streptomycin (BioWhittaker). For PSM imaging, the cells
were harvested at 75% confluence, diluted, and transferred to the
surface of a preassembled, poly-l -lysine treated flow channel
top piece (Figure S1 ).
Type Culture Collection. The cells were cultured in Dulbecco’s
Modified Eagle Medium (DMEM; Lonza) in a humidified incubator at 37
°C with 5% CO2. To active the phosphorylation of Raptor,
the cells were cultured in either glucose-free DMEM for 24 h or in
normal DMEM with 1 mM AICAR for 1 h before the experiment. All the
DMEM was supplied with 10% fetal bovine serum (Invitrogen) and 1%
penicillin and streptomycin (BioWhittaker). For PSM imaging, the cells
were harvested at 75% confluence, diluted, and transferred to the
surface of a preassembled, poly-
top piece (
10
Characterization of Immortalized Kidney Cell Lines
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Immortalized embryonic kidney cell lines, 293T/17 and HEK293, were purchased from American Type Culture Collection, ATCC (Manassas, VA, USA). The U937, U937-Mock, and U937-NDRG2 cell lines [26 (link)] were cultured in Roswell Park Memorial Institute (RPMI) -1640 medium containing 10% fetal bovine serum (FBS) (ATCC, Manassas, VA, USA), HEPES (Lonza, Basel, Swiss), β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA), and penicillin and streptomycin (Lonza, Basel, Swiss). The 293T/17 and HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% FBS, penicillin, and streptomycin at 37 °C in 5% CO2. In some experiments, the cells were treated with chemicals As2O3, MG132, 2’,7’-dichlorofluorescin diacetate (DCFH-DA), okadaic acid, SB216763 (Sigma-Aldrich, MO, USA), and z-VAD-fmk (ENZO, Seoul, Korea). The shMcl-1 clones (TRCN0000005514 and TRCN0000005516) were purchased from Sigma-Aldrich.
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