T47D, BT474, MDA-MB-231, ZR-75-1, and MCF7 breast cancer cell lines and an immortalized normal-like breast cell line 184A1 were obtained as a gift from the laboratory of Dr. Dennis J. Slamon (UCLA, Los Angeles, CA). The cell lines were authenticated by DNA short tandem repeat profiling using the Promega GenePrint 10 system, and analysis was done using the GeneMarker HID software and the ATCC database (Table S3 ). Cells were tested for mycoplasma and were made mycoplasma-free using EZKill mycoplasma removal reagent (HiMedia). T47D, BT474, MDA-MB-231, MCF7, and ZR-75-1 cells were cultured as described previously (19 (link), 36 (link)). The immortalized normal-like 184A1 cell line was cultured in DMEM/F-12 medium (HiMedia) supplemented with 28.18 IU of insulin, 20 ng/ml EGF, and 500 ng/ml hydrocortisone. Basal medium was supplemented with 10% (v/v) fetal bovine serum (Gibco), 2.5 mg/ml Amphotericin-B (Abbott), and 1.25 μl/ml gentamycin (Abbott). All of the cells were cultured at 37 °C in a 5% CO2 incubator. The ER/PR/Her2 receptor status of all of the cells was validated by RT-PCR and expression array analysis (Table 1 ).
Gentamycin
Manufactured by Abbott
Gentamycin is a laboratory equipment product manufactured by Abbott. It is an antibiotic agent used for the detection and quantification of gentamicin in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Geneprint 10 system, by Promega (5 mentions)
Ezkill mycoplasma removal reagent, by HiMedia (4 mentions)
Amphotericin b, by Abbott (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dmem f 12 medium, by HiMedia (2 mentions)
Dmem medium, by Thermo Fisher Scientific (2 mentions)
Erlotinib, by Santa Cruz Biotechnology (1 mentions)
Osimertinib, by Selleck Chemicals (1 mentions)
Str profiling kit, by Promega (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by HiMedia (1 mentions)
Nih 3t3 crl 1658, by American Type Culture Collection (1 mentions)
Hypoxanthine, by Merck Group (1 mentions)
Albumax, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Gentamycin, by HiMedia (1 mentions)
8 protocols using gentamycin
1
Characterization of Breast Cancer Cell Lines
2
Cell Culture and EGFR-TKI Treatment
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PC-9 and PC-9R cells were cultured aseptically in RPMI-1640 medium (Gibco, Catalog #23400–021) and A549 cells in DMEM medium (Gibco, Catalog #12800–017) and incubated at 37 °C in a 5% CO2 incubator. The media were supplemented with 10% fetal bovine serum (Catalog 10270–106), 2.5 mg/ml Amphotericin B and 1.5 μl/ml gentamycin (Abbott). The identity of the cells was authenticated using DNA short tandem repeat (STR) profiling kit (Promega, Geneprint 10 system). The cells were routinely tested for mycoplasma and as and when necessary were treated as per the manual of the EZkill mycoplasma elimination kit (HiMedia, Catalog #CK006). EGFR-tyrosine kinase inhibitors erlotinib (Santacruz Biotechnology) and osimertinib (Selleckchem, Catalog #S7297) were used for the experiments. Both PC-9 and PC-9R cells were modified to stably express luciferase by transduction with retroviral construct as described earlier [16] . The EGFR-TKIs were dissolved DMSO for in vitro experiments and 10% DMSO in 1X PBS for in vivo treatments.
3
Tongue Cancer Cell Lines Characterization
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AW13516 and AW8507 tongue cancer cell lines were obtained from Tata Memorial Hospital while CAL27 cells were procured from ATCC. Cells were tested for mycoplasma and were made mycoplasma-free using the EZKill mycoplasma removal reagent (Cat No. CCK006-1, HiMedia). The cell lines were authenticated by DNA short tandem repeat (STR) profiling using Promega Geneprint 10 system in conjugation with GeneMarker HID software tool. All the cell lines (AW13516, AW8507 and CAL27) were cultured in Dulbecco’s Modified Eagle Medium (Cat No. 12800-017, Gibco) at 37 °C in a 5% CO2 incubator. Culture media was supplemented with 10% fetal bovine serum (FBS) (Cat No. 10270106, Gibco) and 1.25 µL/mL gentamycin (Abbott). Trypsinization was performed using 0.25% Trypsin-EDTA (Cat No. TC245, HiMedia) and cells were suspended in 90% FBS and 10% DMSO (Cat No. D5879, Sigma-Aldrich) freezing mix for long term storage in liquid Nitrogen.
4
Breast Cancer Cell Line Characterization
5
Authenticated Breast Cancer Cell Lines
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T47D, BT474, MDA-MB-231 and MCF7 breast cancer cell lines were obtained from Dr. Slamon's laboratory (Department of Medicine, UCLA, USA). Human embryonic kidney 293FT cells were obtained from Invitrogen. The cell lines were authenticated by DNA Short Tandem Repeat (STR) profiling using the Promega GenePrint 10 system and the analysis was performed using the GeneMarker HID software and the ATCC database. Cells in culture were tested for mycoplasma and were made mycoplasma-free using EZKill Mycoplasma Removal reagent (HiMedia). All the cell lines were grown in DMEM medium (Gibco) supplemented with 10% (v/v) FBS (Gibco), 2.5 mg/ml Amphotericin-B (Abbott) and 1.25 µl/ml Gentamycin (Abbott) and were cultured at 37°C in a 5% CO2 incubator. The ER/PR/Her2 receptor status of breast cancer cells was validated by RT-PCR.
6
Characterization of HNSCC Cell Lines
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AW1351652 (link), AW850752 (link), NT-8e53 (link), and OT953 (link),54 (link) head and neck cancer (HNSCC) cell lines55 (link),56 (link) were obtained from Tata Memorial Hospital (Mumbai), while the NIH/3T3 (CRL-1658) cell line was procured from the American Type Culture Collection (ATCC). The cell lines were authenticated by DNA short tandem repeat (STR) profiling using the Promega Geneprint 10 system in conjugation with the GeneMarker HID software tool. HNSCC cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (cat. no. 12800 – 017; Gibco), supplemented with 10% fetal bovine serum (FBS) (cat. no. 10270106; Gibco) and 1.25 µl/ml gentamycin (Abbott). NIH/3T3 cells were cultured in DMEM supplemented with 10% bovine calf serum (BCS) (cat. no. SH30073.03; Cytiva HyClone) and 1.25 µl/ml gentamycin. Cells were tested for mycoplasma and found to be negative, but as a standard lab protocol, we treated the cells using EZKill Mycoplasma Removal Reagent (cat. no. CCK006 – 1; HiMedia) every 6 months.
7
In vitro Cultivation of Plasmodium falciparum
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Plasmodium falciparum 3D7 strain was cultured in vitro in RPMI 1640 (Life Technologies) supplemented with 10 % human plasma or 0.5 % albumax (Life Technologies), 48 mg L−1of hypoxanthine (Sigma-Aldrich), 2 mg ml−1of sodium bicarbonate (Sigma-Aldrich) and 2 mg ml−1 glucose (Sigma-Aldrich) containing 50 µg ml−1 of gentamycin (Abbott). A haematocrit of 3 % was maintained using human RBCs. Parasites were synchronized with 5 % sorbitol whenever necessary. Fresh RBCs were collected from volunteers after approval from the Institute Ethics Committee of IIT Bombay.
8
Isolation and Culture of Mouse Cortical Neurons
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All the procedures were carried out in aseptic conditions in laminar airflow. The prefrontal cerebral cortices were dissected from mouse E17 embryos under a stereoscopic dissecting microscope in chilled PBS (pH 7.4) containing 40 μg/ml gentamycin. The pieces of tissue were minced in Ca-Mg free Hank's Balanced Salt Solution (Himedia, TL1108) containing 40 μg/ml gentamycin. The minced content was incubated with 0.25% trypsin for 10 min at 37 °C. Trypsin activity was terminated by 1X soybean trypsin inhibitor (Himedia, TCL068) at 37 °C for 5 min. The neurons were dissociated from trypsinized prefrontal cerebral cortices by trituration with fire-polished Pasteur pipette. After viability testing by trypan blue dye exclusion method, 50,000 live cells per coverslip were seeded on poly-L-lysine coated 18 mm glass coverslips. The cells were incubated at 37 °C and 5% CO 2 for 30 min in the CO 2 incubator (New Brunswick an Eppendorf company, Galaxy 48R, 41823) for adhesion. Four coverslips were placed in a 60 mm tissue culture dish. The cells were fed with neurobasal medium (Gibco, 15630-106) supplemented with 0.2% B-27 (Gibco, 17504-044), 40 μg/ml gentamycin (Abbott), 25 mM HEPES (Himedia, TL1108) and 1% Glutamax (Gibco, 35050-061). The culture was maintained at 37 °C and 5% CO 2 in the CO 2 incubator. Two-third of the old culture medium was replaced with fresh medium on every third day.
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