Cytotoxicity evaluation was performed using normal human skin fibroblast (BJ cell line, ATCC® CRL-2522TM). In turn, anti-proliferative activity was assessed towards cancer cells: human lung adenocarcinoma (A549, ATCC® CCL-185TM), human hepatocellular carcinoma (HepG2 cell line, ATCC® HB-8065TM), and human breast adenocarcinoma (MCF-7, ATCC® HTB-22TM) as well as towards normal cells (BJ cell line was used as model of normal cells). All cell lines were purchased from ATCC, United Kingdom and cultured according to manufacturer recommendations.
Bj cell line
Manufactured by American Type Culture Collection
Sourced in United States, United Kingdom
The BJ cell line is a continuous cell line derived from normal human foreskin fibroblasts. It is a commonly used model system for the study of cellular senescence and immortalization processes.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Merck Group (3 mentions)
Streptomycin, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by PAN Biotech (3 mentions)
Penicillin streptomycin, by Euroclone (2 mentions)
Ccl 185tm, by American Type Culture Collection (1 mentions)
Crl 2522tm, by American Type Culture Collection (1 mentions)
Htb 22tm, by American Type Culture Collection (1 mentions)
Hb 8065tm, by American Type Culture Collection (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Hyclone, by GE Healthcare (1 mentions)
Heraeus cytoperm 2, by Thermo Fisher Scientific (1 mentions)
Crl 2522, by American Type Culture Collection (1 mentions)
Crl 1721, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Euroclone (1 mentions)
Pd98059, by Promega (1 mentions)
Ht1080 fibrosarcoma cells, by American Type Culture Collection (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Pin tool, by V&P Scientific (1 mentions)
Celltiter glo, by Promega (1 mentions)
12 protocols using bj cell line
1
Cytotoxicity and Anti-proliferative Evaluation
2
Human Fibroblast Cell Culture Protocol
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Human fibroblast (BJ) cell line was purchased from ATCC (Manassas, Virginia, USA). The cells were maintained in DMEM F12 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) in the plastic tissue culture flasks (Falcon, GE Healthcare Life Sciences, Chicago, Illinois, USA) at 37 °C in a humidified atmosphere containing 5%, CO2 and 95% air. Both mediums were supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone, GE Healthcare Life Sciences, Chicago, Illinois, USA) contained 1% antibiotic streptomycin-penicillin (Thermo Fisher Scientific, Waltham, MA, USA).
3
Evaluating Wound Healing Properties of Biomaterials
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The cell culture experiments were performed using normal human skin fibroblasts, i.e., BJ cell line (ATCC, London, UK), as it is known as a good model for the evaluation of wound healing in vitro [33 (link),38 (link),39 (link),40 (link),41 ]. The cells were grown in EMEM medium with an addition of 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. According to ATCC directions, BJ cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 and 95% air (Heraeus cytoperm 2, Thermo Scientific, Waltham, MA, USA). To assess fibroblast response to the tested biomaterials, the liquid extracts from the samples of curdlan-based biomaterials and KALTOSTAT® specimens were prepared according to ISO 10993-5:2009 standard directions [37 ], as described in Section 2.8 . For evaluation of cell viability, extracts were prepared using EMEM supplemented with 2% FBS, while extracts obtained in EMEM with an addition of 10% FBS was applied for estimation of cell proliferation. As a control extract, appropriate EMEM medium (with 2% or 10% FBS) incubated without biomaterials was utilized. The ISO 10993-5:2009 standard guidelines [37 ] are commonly applied for the evaluation of biological properties in vitro of biomaterials with biomedical potential [33 (link),34 (link),42 (link),43 (link),44 (link)].
4
Modeling melanoma and neuroendocrine tumor cells
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The rat pheochromocytoma cell line PC12 (ATCC® CRL-1721™) [47 (link)] together with the two human malignant melanoma cell lines LCP-mel (RRID:CVCL_7053) and PNP-mel (RRID:CVCL_G320) [48 (link)] have been used as experimental models for our study. The last are primary human melanoma cells derived from tumor biopsies and were kindly donated from the Istituto Dermopatico dell’Immacolata (IDI, Rome, Italy). As a non-tumor control, we used a human fibroblast cell line from a healthy donor, the BJ cell line (CRL-2522™- ATCC®, Manassas, VA, USA). The mouse melanoma cell line B16F10 (ATCC® CRL-6223™ ATCC®, Manassas, VA, USA) was also used, only for the melanin content assays.
Human malignant melanoma cell lines were cultured in RPMI medium with stable glutamine, supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (1 U/mL) (all from Euroclone®, Pero, Italy), (complete medium), in a humidified atmosphere with 5% CO2, at 37 °C.
PC12 cells (passage 12–25) were maintained in atmosphere of 5% CO2/95% humidified air at a fixed temperature of 37 °C and cultured in 60 mm Ø plastic plates with Dulbecco’s modified Eagle’s medium (DMEM/F12) added with 10% Horse Serum (HS), 5% Fetal Bovine Serum (FBS) and 1% of penicillin/streptomycin.
Human malignant melanoma cell lines were cultured in RPMI medium with stable glutamine, supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (1 U/mL) (all from Euroclone®, Pero, Italy), (complete medium), in a humidified atmosphere with 5% CO2, at 37 °C.
PC12 cells (passage 12–25) were maintained in atmosphere of 5% CO2/95% humidified air at a fixed temperature of 37 °C and cultured in 60 mm Ø plastic plates with Dulbecco’s modified Eagle’s medium (DMEM/F12) added with 10% Horse Serum (HS), 5% Fetal Bovine Serum (FBS) and 1% of penicillin/streptomycin.
5
Malignant Melanoma Cell Lines for Research
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Five malignant melanoma (MM) cell lines were used as a multiple experimental model for our study. They were chosen among 27 collected in our laboratory as the most suitable to represent the different molecular types of melanoma [34 (link)]. They were all obtained from the Istituto Dermopatico dell’Immacolata (IDI, Rome, Italy) and are listed below:
All cell lines were cultured in RPMI medium with stable glutamine, supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (1 U / mL) (all components from Euroclone, Pero, Italy) (complete medium), in a humidified atmosphere with 5% CO2 at 37 °C.
CN-mel (CN), derived from melanoma lymph node metastasis, carrying a NRASQ61R gene mutation;
PR-mel (PR), derived from melanoma cutaneous metastasis, carrying a BRAFV600R gene mutation;
A375, derived from melanoma cutaneous metastasis, carrying CDKN2AE61*-E69* gene mutations and a BRAFV600E gene mutation;
SK-mel (SK), derived from melanoma lymph node metastasis, carrying a PTENT167A gene mutation;
GR-mel (GR), derived from a primary melanoma, carrying a homozygous deletion in the CDKN2B gene.
All cell lines were cultured in RPMI medium with stable glutamine, supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (1 U / mL) (all components from Euroclone, Pero, Italy) (complete medium), in a humidified atmosphere with 5% CO2 at 37 °C.
6
Cell Culture and MAPK Modulation Techniques
7
Cell Viability and Cytotoxicity Evaluation
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The BJ cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured according to recommendations. Cell culture media were purchased from ATCC. Cells were routinely tested for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza). Cells were grown to 80% confluence, collected, and plated in 25 μL of medium per well in 384-well plates (Costar 3712). Compounds were diluted as described above and transferred to cells using a pin tool (V&P Scientific) equipped with FP1S50 pins resulting in final compound concentrations of 25 μM, and the plates incubated for 72 h at 37°C in 5% CO2. CellTiter-Glo (Promega) detection reagent was added following the manufacturer’s instructions, and luminescence was measured using an EnVision (PerkinElmer) plate reader. Data were log transformed and EC50 values were determined using GraphPad Prism 6 (GraphPad Software).
Cytotoxicity of compounds to J774.A1 macrophages was determined by dose-response curves as described previously [43 (link)]. In the absence of growth inhibitors or DMSO, the macrophages increased in number ~6-fold over 96 h in Minimum Essential Medium, employed for both macrophage infections and the toxicity assays.
Cytotoxicity of compounds to J774.A1 macrophages was determined by dose-response curves as described previously [43 (link)]. In the absence of growth inhibitors or DMSO, the macrophages increased in number ~6-fold over 96 h in Minimum Essential Medium, employed for both macrophage infections and the toxicity assays.
8
Culturing Mammalian Cell Lines
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The mouse monocyte/macrophage-like cell line RAW 264.7 and human foreskin fibroblasts (BJ cell line) were obtained from the American Type Culture Collection. The RAW 264.7 and BJ cells were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) and Minimum Essential Medium (Eagle) [MEM(E)] (HiMedia Laboratories, LLC) media, respectively. The culture media were supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 100 units of penicillin/100 µg/ml streptomycin (HiMedia Laboratories, LLC), and the cells were grown in a humidified incubator in an atmosphere with 5% CO2 at 37˚C.
9
Culturing Human Skin Fibroblasts
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Normal human skin fibroblasts (BJ cell line) obtained from American Type Culture Collection (ATCC, Teddington, UK) were used in the presented work. Human skin fibroblasts were cultured in a dedicated Eagle’s Minimum Essential Medium (EMEM, ATCC-LGC Standards, Teddington, UK). The culture medium also contained: 10% fetal bovine serum (FBS, Pan-Biotech GmbH, Aidenbach, Bavaria, Germany), streptomycin (100 µg/mL), and penicillin (100 U/mL), which were obtained from Sigma-Aldrich Chemicals (Warsaw, Poland). The cell line was cultured in the recommended by ATCC conditions: 37 °C, 5% CO2, and 95% air humidity.
10
Normal Skin Fibroblast Cultivation
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Normal human skin fibroblasts (BJ cell line) were obtained from American Type Culture Collection ATCC-LGC Standards (Teddington, UK). BJ cells were cultured in a dedicated Eagle’s Minimum Essential Medium (EMEM, ATCC-LGC Standards, Teddington, UK) supplemented with 10% FBS (FBS, Pan-Biotech GmbH, Aidenbach, Bavaria, Germany), 100 μg/mL streptomycin, and 100 U/mL penicillin (Sigma-Aldrich Chemicals, Warsaw, Poland). The cells were maintained under standard conditions at 37 °C in a humidified atmosphere of 5% CO2 and 95% air.
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