UACC-812 and MDA-MB-415 cells were purchased from ATCC (Manassas, VA). RS4.11 and HL-60 cell lines were obtained from Ashish Kumar lab (CCHMC). All cell lines were authenticated by STR profiling at Genetica (Burlington, NC). UACC-812 cells were grown in Leibovitz’s L-15 (Gibco) medium with 2 mM l -glutamine containing 20% fetal bovine serum (FBS) and 0.1% antibiotic and antimycotic (Gibco). MDA-MB-415 cells were grown in Leibovitz’s L-15 (Gibco) medium with 2 mM l -glutamine supplemented with 10 μg/ml insulin (Sigma), 10 μg/ml glutathione (Calbiochem), 15% FBS and 0.1% antibiotic and antimycotic (Gibco). SKBR3, BT474, MDA-MB-231, CAL51, T47D cells were cultured in RPMI 1640 (Gibco) containing 10% FBS with 0.1% antibiotic and antimycotic (Gibco). MDA-MB-453 cells were cultured in improved minimum essential medium (Gibco) containing 20% FBS with 0.1% antibiotic and antimycotic (Gibco). All cells were cultured in a humidified atmosphere in 5% CO2 at 37 °C. All cells were regularly tested for mycoplasma contamination and were negative.
Leibovitz s l 15
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Leibovitz's L-15 is a cell culture medium developed by American biochemist John E. Leibovitz. It is a commonly used medium for the in vitro cultivation of various cell types, particularly those that prefer low oxygen conditions.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (35 mentions)
Rpmi 1640, by Thermo Fisher Scientific (18 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (15 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (14 mentions)
Mda mb 231, by Thermo Fisher Scientific (12 mentions)
Penicillin, by Thermo Fisher Scientific (11 mentions)
Streptomycin, by Thermo Fisher Scientific (10 mentions)
Trypsin edta, by Thermo Fisher Scientific (10 mentions)
Mccoy s 5a, by Thermo Fisher Scientific (9 mentions)
Mcf 7, by Thermo Fisher Scientific (9 mentions)
Softworx, by GE Healthcare (9 mentions)
Mda mb 231, by American Type Culture Collection (6 mentions)
Mcf 7, by American Type Culture Collection (6 mentions)
Hct116, by Thermo Fisher Scientific (5 mentions)
Sw620, by Thermo Fisher Scientific (5 mentions)
Mda mb 453, by Thermo Fisher Scientific (5 mentions)
L glutamine, by Thermo Fisher Scientific (5 mentions)
Dmem f12, by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Merck Group (5 mentions)
Deltavision elite system, by GE Healthcare (5 mentions)
97 protocols using leibovitz s l 15
1
Cell Line Culturing and Maintenance
2
Culture Protocols for Cancer Cell Lines
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The human breast cancer cell line, MDA-MB-231, the human gastric cancer cell line, SNU-16, and the mouse colon carcinoma cell line CT26.WT were purchased from the American Type Culture Collection. The human gastric cancer cell line 44As3 was obtained from Yasuda Women's University.43 (link) MDA-MB-231 cells were cultured in Leibovitz's L-15 (Thermo Fisher Scientific) or RPMI1640 (Thermo Fisher Scientific) both supplemented with 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific) at 37°C in a humidified atmosphere without CO2 (Leibovitz's L-15) or in a humidified atmosphere of 5% CO2 (RPMI1640). SNU-16, CT26.WT, and 44As3 cells were cultured in RPMI1640 supplemented with 10% heat-inactivated FBS at 37°C in a humidified atmosphere of 5% CO2. The 293F cells (Thermo Fisher Scientific, R790-07) were cultured in FreeStyle 293 Expression Medium (Thermo Fisher Scientific, 12338-018) at 37°C in a humidified atmosphere of 8% CO2 with shaking at 135 rpm.
3
Zebrafish Liver Cell Line Cultivation
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Zebrafish liver cell line (ZFL) was kindly provided by the China Zebrafish Resource Center (CZRC catalog ID: Cell2, http://zfish.cn/Products/ProductDetail.aspx?CZRCID=Cell2 , access date: 13 October 2020) and stored in the Preservation and Research Center for Aquatic Animal Cells, Huazhong Agriculture University. ZFL grew in a mixed medium consisting of 48% Leibovitz’s L-15, 15% Ham’s F-12, 32% Dulbecco’s modified Eagle medium (Gibco, Carlsbad, CA, USA), 4% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA), and 1% antibiotics (50 U/mL anti-penicillin and 50 μg/mL anti-streptomycin) (Gibco, Carlsbad, CA, USA). The cells were cultured in a cell culture flask (base area: 25 cm2) in a conventionally humidified incubator at 28 ℃, 5% CO2, and 4 mL fresh medium was replaced at intervals of one day. When the monolayer cell density was greater than 90%, the cells were digested with 0.25% trypsin and cell passage cultivation was carried out.
4
Cultivation of human cancer cell lines
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Human colon cancer cell lines, LoVo, HCT116, HT29, SW480 and the human T-lymphocyte cell line Jurkat were obtained from the Cell Bank, Chinese Academy of Science. Cells were routinely grown and passaged as previously described [16 (link), 21 (link)]. In brief, cells were grown in F12K (Gibco, Grand Island, NY, USA) (LoVo), McCoy's 5A (Gibco) (HCT116 and HT29), Leibovitz's L-15 (Gibco) (SW480), or RPMI-1640 (Gibco) (Jurkat) supplemented with 100 mL/L fetal bovine serum (FBS) (Gibco), 100,000 IU/L penicillin, and 100 mg/L streptomycin (Gibco) under a humidified atmosphere of 5% CO2 at 37°C.
5
Juvenile Greenlip Abalone Farming and Analysis
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Juvenile greenlip abalone (Haliotis laevigata) were farmed in Tasmania and distressed for a week without feeding in 4000-l tanks filled with filtered sea water at 14–18 °C. Dispase, sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl2), CelLytic M reagent, ethidium homodimer, phenazine methosulfate (PMS), 3,3′,5,5′ tetramethylbenzidine (TMB), bovine serum albumin (BSA) and ammonium sulphate were purchased from Sigma-Aldrich, Australia. Phosphate buffer solution (PBS pH 7.4), antibiotic–antimycotic solution (anti-anti) 100 × , fetal bovine serum, KnockOut™ serum replacement, minimum essential medium (MEM) vitamin solution 100 × , minimum essential medium (MEM) amino acid solution 100 × , chemically defined lipid concentrate, (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) assay kit, Insulin-Transferrin-Selenium-Ethanolamine (ITS -X) (100 ×), fluorescein isothiocyanate (FITC)-conjugated anti-rabbit antibody from goat and Qubit 2.0 Fluorometric Protein Quantification kit were supplied by Invitrogen (US). F-12 nutrient mixture ham medium, Leibovitz’s L-15, modified Eagle’s medium (MEM) and Dulbecco’s modified Eagle’s medium (DMEM) were supplied by Gibco, Lipimax® by Selbourne Biological Service (Aus), magnesium chloride and magnesium phosphate by MERCK. Rabbit anti-hemocyanin polyclonal antibody was produced in-house.
6
Quantifying mitotic dynamics in CRISPRi/a cells
7
Breast Cancer Cell Line Culture Protocols
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Breast cancer cell lines BT-549, MDA-MB-231, MDA-MB-468, and MCF-7 as well as an immortalized mammary epithelial-like cell line, MCF-10A were obtained from Procell biological company (Shanghai, China). BT-549 and MCF-7 cells were cultured in RPMI-1640 (Procell) supplemented with 10% (v/v) fetal bovine serum (FBS), 0.01 mg/mL insulin (Procell), 2 mM l -glutamine, 0.1 mg/mL streptomycin, and 100 U/mL penicillin at 37 °C in a humidified atmosphere containing 5% CO2. MDA-MB-231 and MDA-MB-468 cells were cultured in Leibovitz's L-15 (Gibco) with 10% (v/v) FBS, 2 mM l -glutamine, 0.1 mg/mL streptomycin, and 100 U/mL penicillin at 37 °C in a standard humidity incubator. MCF-10A was cultured in DMEM (Procell) supplemented with 5% horse serum, 20 ng/mL epidermal growth factor, 0.5 μg/mL hydrocortisone, 0.1 mg/mL streptomycin, 100 U/mL penicillin, and 0.01 mg/mL insulin at 37 °C in a humified atmosphere containing 5% CO2.
8
Cell Line Culture Conditions
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All cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). MCF10A cells were grown in DMEM/F12 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% horse serum (Invitrogen), EGF (ProSpec, Rehovot, Israel), hydrocortisone (Sigma, Beijing, China), insulin (Sigma), and 1% penicillin–streptomycin (Invitrogen). MDA-MB-231, MCF7, and T47D cells were grown in DMEM (Invitrogen). MDA-MB-468 and SK-BR-3 cells were cultured with Leibovitz’s L-15 (Gibco, Grand Island, USA) and McCoy’s 5A (Gibco), respectively. Except for MDF10A medium, all growth media were supplemented with 10% fetal bovine serum (Invitrogen), 100 Uml−1 penicillin, and 100 μgml−1 streptomycin (Invitrogen). MDA-MB-468 cells were cultured in a humidified atmosphere of 0.03% CO2 at 37°C while all other cell lines were cultured in the same conditions except that the CO2 concentration was 5%.
9
EMT Induction in Breast Cancer Cell Lines
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T47D, MCF7, ZR-75-1, BT474, MDA-MB-231, MDA-MB-435S, and BT549 cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). MCF-10A cells were obtained from American Tissue Culture Collection (ATCC, Manassas, VA, USA). T47D, ZR-75-1, BT474, MDA-MB-231, and BT549 cells were maintained in RPMI 1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 IU ml−1 penicillin, 100 μg ml−1 streptomycin, and 2 mM L -glutamine. MCF7 cells were maintained in DMEM (Gibco) supplemented with 10% FBS, 100 IU ml−1 penicillin, 100 μg ml−1 streptomycin, and 2 mM L -glutamine; MDA-MB-435S cells were maintained in Leibovitz's L-15 (Gibco) supplemented with 10% FBS; MCF-10A cells were maintained in DMEM/F12 (1 : 1) (HyClone, Logan, UT, USA) supplemented with 5% horse serum (HyClone), 20 ng ml−1 epidermal growth factor (EGF; Peprotech, Rocky Hill, NJ, USA), 100 ng ml−1 cholera toxin (Sigma, St Louis, MO, USA), 10 μg ml−1 insulin (Sigma), and 0.5 μg ml−1 hydrocortisone (Sigma).
For EMT induction, cells were cultured in media with 50 ng ml−1 IL-6 (Peprotech), 20 ng ml−1 EGF (Peprotech) and 20 ng ml−1 bFGF (Peprotech), or 10 ng ml−1 transforming growth factor-β1 (TGF-β1; Peprotech) at 37 °C in 5% CO2.
For EMT induction, cells were cultured in media with 50 ng ml−1 IL-6 (Peprotech), 20 ng ml−1 EGF (Peprotech) and 20 ng ml−1 bFGF (Peprotech), or 10 ng ml−1 transforming growth factor-β1 (TGF-β1; Peprotech) at 37 °C in 5% CO2.
10
Lymnaea CNS Neurite Sprouting Assay
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Isolated Lymnaea CNS were incubated in 3 mL of defined medium (DM; comprised of 50% Leibovitz’s L-15 (Gibco, Dublin, Ireland) and additional salts) containing 10−5 M RA (Sigma, Oakville, ON, Canada) (to induce neural regeneration/neurite sprouting) in a plastic Falcon dish (VWR, Radnor, PA, USA) for 72 h. Different CNS were also incubated in 0.1% EtOH (Greenfield Global, Brampton, ON, Canada) as a vehicle control. Following the 72 h incubation period, any neurite sprouting from individual nerves emanating from the CNS were imaged using Q-Capture imaging software (v2.90.1, Q Imaging, Surrey, BC, Canada).
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