Human intestinal Caco-2 cells (purchased from the American Type Culture Collection–ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, Inc., St. Louis, MO, USA) supplemented with 20% (v/v) fetal bovine serum (FBS; Biological Industries, Beit Haemek, Israel) and 1% (v/v) penicillin-streptomycin solution (Biological Industries). The human cervical cancer HeLa cell line (courtesy of Prof. Nahum Shpigel, The Hebrew University of Jerusalem, Israel) was cultured in DMEM medium (Sigma-Aldrich) supplemented with 10% (v/v) FBS (Biological Industries) and 1% (v/v) penicillin-streptomycin solution (Biological Industries). The rat intestinal epithelium IEC-18 cell line (courtesy of Prof. Betty Schwartz, The Hebrew University of Jerusalem, Israel) was cultured in DMEM (Sigma-Aldrich) supplemented with 10% (v/v) FBS (SAFC Biosciences Lenexa, KS, USA), 1% (v/v) L-glutamine (Biological Industries, Beit Haemek, Israel), and 1% (v/v) penicillin-streptomycin solution (Biological Industries). All cells were grown under a 37°C humidified atmosphere containing 95% air and 5% CO2.
Penicillin streptomycin solution
Manufactured by Sartorius
Sourced in Israel, United States, United Kingdom
Penicillin-streptomycin solution is a cell culture media supplement used in laboratory settings. It contains the antibiotics penicillin and streptomycin, which inhibit the growth of bacteria and other microorganisms. The solution is commonly used to prevent bacterial contamination in cell culture experiments.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Sartorius (29 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (15 mentions)
L glutamine, by Sartorius (9 mentions)
Rpmi 1640 medium, by Sartorius (8 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (7 mentions)
Fetal calf serum (fcs), by Sartorius (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (5 mentions)
Sodium pyruvate, by Sartorius (5 mentions)
Non essential amino acids (neaa), by Sartorius (4 mentions)
Glutamine, by Sartorius (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Trypsin edta, by Sartorius (3 mentions)
Blasticidin, by Thermo Fisher Scientific (3 mentions)
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Trypsin, by Sartorius (3 mentions)
Rpmi 1640, by Sartorius (3 mentions)
Dulbecco modified eagle medium (dmem), by Sartorius (3 mentions)
Fetal bovine serum (fbs), by Cytiva (2 mentions)
Dmem medium, by Thermo Fisher Scientific (2 mentions)
90 protocols using penicillin streptomycin solution
1
Cell Culture Protocols for Cell Lines
2
Cultivation of K562 and HEK293T Cell Lines
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The following cell lines were used in the current study: K562 chronic myeloid leukemia cells and HEK293T human embryonic kidney cells. K562 cells were maintained in RPMI-1640 medium containing 1% l -glutamine and 25 mM HEPES pH 7.4 (Gibco, Waltham, MA, USA), supplemented with 10% fetal calf serum (FCS; Gibco) and 1% penicillin–streptomycin solution (Sartorius, Goettingen, Germany) and passaged every 2–4 days. HEK293T cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Rhenium, Modiin, Israel) supplemented with 10% FCS, 1% penicillin–streptomycin solution, and 1% l -glutamine (Sartorius) and passaged every 2–4 days.
3
Maintenance and Manipulation of Human Cell Lines
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Human U2OS cells were maintained in low glucose DMEM (Biological Industries, Beit-Haemek, Israel), supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT), 1% Glutamine (Biological Industries, Beit Haemek, Israel), and 1% penicillin–streptomycin solution (Biological Industries). MCF7 and HeLa cells were maintained in high glucose DMEM (Biological Industries), supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin solution. All cells were grown at 37 °C in 5% CO2. The GFP-dystrophin-MS2 U2OS cell line22 (link) was selected using 165 μg/μl hygromycin (Sigma-Aldrich) and activated using Ponasterone A (PonA) at 5 μg/μl for 24 h (unless otherwise specified). Hyper-osmolarity treatment was performed with 1:100 10XPBS for 10 min. ATP depletion was performed using either 2-deoxy-d -glucose (20 mM, Sigma) or Na-azide (20 mM, Sigma) in medium for either 15 min or 1 h. Hoechst 33342 (2.5 μM) was used for labeling DNA. Puromycin (Invivogen) was used at a concentration of 10 μg/ml for 30 min in medium. Cycloheximide (Sigma-Aldrich) was used at a concentration of 100 μg/ml for 4 h in medium.
4
Cell Culture Protocol for Skin Cancer
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Human keratinocytes HaCaT, human melanoma cells A375 and murine melanoma cells B16 were purchased from the National Collection of Authenticated Cell Cultures. The above cells were cultured in DMEM medium (Gibco) with 10% FBS (Bioind) and 1% penicillin–streptomycin solution (Bioind) at 37 ℃ incubator supplemented with 5% CO2. Cell lines were tested monthly for mycoplasma and validated via STR testing.
5
Modulating RAC1 in Lung Cancer Cells
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A549, PC9, H1299, H460, and HBE cells were maintained in RPMI-1640 medium supplemented with 10% FBS and 100 U/ml penicillin-streptomycin solution (Bioind, Israel) in 5% CO2 incubator. CMV-RAC1, CMV-sh-RAC1 and their controls plasmids were purchased from Shanghai Genechem Co., LTD. A549 and PC9 Cells transfected using Lipofectamine™3000 (Invitrogen, Waltham City, Massachusetts, USA) with vector, RAC1, sh-control and sh-RAC1 plasmids followed by 500 μg/ml G418 screening were pooled collected and positive clones were selected and identified for stably expressing RAC1 or silencing RAC1. For experiments involving IR exposure, growing cells with serum-free culture medium after indicated dose of 2 Gy/min were continuously incubated with 10% FBS at 37°C for 48 h prior to analysis.
6
Ovariectomy in Sprague Dawley Rats
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Female 8-week-old specific pathogen-free Sprague Dawley (SD) rats were randomly divided into the OVX group (n = 20) and the SHAM group (n = 20). After acclimatization for 1 week, the rats in the OVX group underwent bilateral OVX, while similar sizes of adipose tissue near the ovaries were resected in the SHAM group. All rats were housed under standard temperature and humidity, with a 12/12-h light/dark cycle and free rodent diet.
After 12 weeks, all rats were euthanized. BMSCs were aseptically harvested from bilateral femurs and tibiae and then incubated using Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F12, Hyclone, UT, USA) supplemented with 10% fetal bovine serum (FBS; BioInd, Kibbutz Beit Haemek, Israel) and 1% penicillin–streptomycin solution (BioInd) in a 5% CO2 humidified atmosphere at 37 °C. Only third-generation BMSCs were used for RNA-seq. For further verification experiments, the third–fifth-generation BMSCs collected from 3 to 4-week-old female SD rats were used.
After 12 weeks, all rats were euthanized. BMSCs were aseptically harvested from bilateral femurs and tibiae and then incubated using Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F12, Hyclone, UT, USA) supplemented with 10% fetal bovine serum (FBS; BioInd, Kibbutz Beit Haemek, Israel) and 1% penicillin–streptomycin solution (BioInd) in a 5% CO2 humidified atmosphere at 37 °C. Only third-generation BMSCs were used for RNA-seq. For further verification experiments, the third–fifth-generation BMSCs collected from 3 to 4-week-old female SD rats were used.
7
Cell Culture and miRNA Transfection
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HeLa and SiHa cells were obtained from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China) and cultured in RPMI 1640 medium (BioInd, Kibbutz Beit Haemek, Israel) containing 10% foetal bovine serum (FBS, BioInd) and 1% penicillin-streptomycin solution (BioInd) at 37°C and 5% CO2. Chemosynthetic hsa-miR-195-5p mimics, mimics control, hsa-miR-195-5p inhibitor, inhibitor control, ATG9A overexpression plasmid, and ATG9A negative control were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). HeLa and SiHa cells were seeded at 80%–90% density and transfected with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
8
Investigating miR-195-5p in Cervical Cancer
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Human cervical cancer cells HeLa and SiHa were purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China) and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (BioInd, Kibbutz Beit Haemek, Israel) supplemented with 10% fetal bovine serum (FBS) (BioInd) and 1% penicillin-streptomycin solution (BioInd) at 37°C, 5% CO2, and 50% relative humidity. The medium was replaced according to the growth rate of the cells. Chemosynthetic Has-miR-195-5p mimics, negative control (NC), Has-miR-195-5p inhibitor, and NC inhibitor were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). Cells were seeded at 70–90% density and transfected with LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
9
Isolation and Culture of Rat BMSCs
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Bilateral femurs and tibiae collected from OVX rats and SHAM rats were dissected free of soft tissue under aseptic conditions. Then both ends of the bones were removed with scissors, and a syringe needle was inserted into the medullary cavity. All the bone marrows were flushed out using Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12, Hyclone, UT, USA) supplemented with 10% fetal bovine serum (FBS; BioInd, Kibbutz Beit Haemek, Israel) and 1% penicillin-streptomycin solution (BioInd) into cell culture dishes. The marrow mixture provided suitable environment for BMSCs culture. The cells were incubated in 5% CO2 humidified atmosphere at 37°C. The culture medium was replaced every 2 days. When cells density reached 80%, adherent cells were digested by 0.25% trypsin (KeyGen, Nanjing, China) and passaged into new dishes.
10
Doxorubicin Cytotoxicity Assay
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DOX solution was obtained from EBEWE Arzneimittel GmbH, Vienna, Austria and kept at −20 °C. RPMI 1640 medium, minimum essential medium (MEM), fetal bovine serum (FBS), non-essential amino acids, antibiotic–antimycotic solution, penicillin–streptomycin solution, l -glutamine, and trypsin/EDTA were purchased from Bioind, Beit Haemek, Israel.
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