For this study all cell lines, including human osteosarcoma SJSA-1 (CRL-2098) and the human immortalized osteoblast hFOB 1.19 (ATCC, CRL-11372), were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). SJSA-1 and hFOB 1.19 were grown in a humidified chamber containing 5% CO2 and cultured in a growth medium (HyClone™ MEM-α medium, Marlborough, MA, USA, SH30265FS) containing 10% fetal bovine serum (Fisher Scientific, Waltham, MA, USA, ES009B) and 1% HyClone™ penicillin-streptomycin (Cytiva, SV30010). However, the hFOB 1.19 cell line media also contained 0.3 mg/mL G418 (Fisher Scientific, 10-131-035) and was maintained at 34 °C, as per ATCC protocol. Cells in 100 mm tissue culture dishes (Fisher Scientific, 430167) were treated with 1 μM 4SC-202 (Adooq Bioscience, Irvine, CA, USA, A14354-25) for 24 h unless differently stated. The corresponding amount of DMSO (vehicle) was used as a control. All assays were performed at 37 °C.
Hfob1
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The HFOB1.19 is a laboratory equipment designed for specific functions. It is a high-precision instrument used for analytical purposes in various scientific and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Hfob1, by American Type Culture Collection (24 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (22 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (15 mentions)
Dmem f12, by Thermo Fisher Scientific (14 mentions)
Saos 2, by American Type Culture Collection (12 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (9 mentions)
Mccoy s 5a medium, by Thermo Fisher Scientific (8 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (7 mentions)
Hfob1, by National Collection of Authenticated Cell Cultures (7 mentions)
Mg 63, by American Type Culture Collection (6 mentions)
Rpmi 1640, by Thermo Fisher Scientific (6 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (6 mentions)
Saos 2, by Thermo Fisher Scientific (6 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Mnng hos, by Thermo Fisher Scientific (5 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (3 mentions)
50 protocols using hfob1
1
Evaluating 4SC-202 Effects on Osteosarcoma
2
Osteosarcoma Cell Line Cultivation and Transfection
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The human osteosarcoma cell lines U2-OS, MG63, HOS and SAOS-2, as well as the normal osteoblastic cell line hFOB 1.19 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). hFOB 1.19 cells were cultured in Dulbeccos modified Eagles medium (DMEM)/F-12 (1:1; Invitrogen) and osteosarcoma cells in osteoblast growth medium (Invitrogen). All cells were maintained at 37°C in a humidified incubator with 5% CO2 in media supplemented with 10% fetal bovine serum (FBS; Invitrogen), 1% of 100 U/ml penicillin and streptomycin (Invitrogen) and 2 mM glutamine. The miR-505 mimic and mimics negative control (miR-NC) were obtained from Guangzhou RiboBio, Co., Ltd. (Guangzhou, China). Cells were transfected with 30 nM miR-505 mimics or miR-NC using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturers instructions. Overexpression of HMGB1 was achieved using the pcDNA3.1/HMGB1 transfection and the pcDNA3.1/CAT (vector) was used as its negative control.
3
Osteosarcoma Cell Line Maintenance and Fractalkine Knockdown
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The human osteosarcoma cell lines MG63, U2OS and the human osteoblast cell line hFOB 1.19 were purchased from the American Type Cell Culture Collection (Manassas, VA, USA). All cell lines were maintained in DMEM supplemented with 20 mM HEPES, 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C with 5% CO2. hFOB 1.19 cells were maintained under the same condition but at 33°C.
To generate fractalkine stable knockdown cell line of MG63 cells, the cells were transfected with fractalkine shRNA by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), and fractalkine shRNA-expressing MG63 cells were selected using puromycin (1 μg/mL). The selected cells were expanded to generate clonal cell populations.
To generate fractalkine stable knockdown cell line of MG63 cells, the cells were transfected with fractalkine shRNA by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), and fractalkine shRNA-expressing MG63 cells were selected using puromycin (1 μg/mL). The selected cells were expanded to generate clonal cell populations.
4
Culturing Human Osteosarcoma Cell Lines
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Human osteosarcoma cell lines (HOS, 143B, and U2OS) and human fetal osteoblast cell line hFOB 1.19 were obtained from the FuHeng Cell Center (Shanghai, China). The osteosarcoma cell lines were authenticated by ShangHai Biowing Applied Biotechnology, performing a STR profiling analysis, as described by Capes-Davis and according to the American National Standards Institute (ANSI) standard (ASN-0002) set forth by the ATCC Standards Development Organization. Mycoplasma testing was performed using the Venor GeM mycoplasma detection kit (Minerva Biolabs, Berlin, Germany). Human cell line hFOB 1.19 was cultured in Dulbecco’s modified Eagle’s medium (DMEM)-F12 medium (Invitrogen, Karlsruhe, Germany), and human osteosarcoma cell lines HOS, 143B, and U2OS were cultured in DMEM (Gibco, Life Technologies, Darmstadt, Germany), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Invitrogen) and 1% (v/v) penicillin/streptomycin (Invitrogen), under adherent conditions at 37°C in a humidified incubator with 5% CO2/95% air.
5
Cultivation of Human Fetal Osteoblasts
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Human fetal osteoblast cell line hFOB 1.19 (CRL-11372) was purchased from American Type Culture Collection (ATCC®) (Manassas, VA 20110). The hFOB 1.19 cells were cultured in Dulbecco’s Modified Eagle Medium F-12 nutrient mixture (DMEM/F12) (Invitrogen GmBH, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (FBS) (Invitrogen GmBH, Karlsruhe, Germany) and 1% penicillin/streptomycin serum (Invitrogen GmBH, Karlsruhe, Germany) [22 (link),52 (link)]. The cells were incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO2 (Sheldon, Cornelius, OR, USA) to provide the optimum temperature, moisture (sterile environment), and pH. The cells were monitored closely for 24 h. All cell culture-related works were conducted in Biosafety Cabinet (BSC) Class II to maintain sterility conditions.
6
Silencing circTUBGCP3 in Osteosarcoma Cell Lines
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Four human osteosarcoma cell lines (143B, HOS, U20S, and MG-63), HEK-293 and human osteoblast cells hFOB1.19 were commercially acquired from the Chinese Academy of Sciences (Shanghai, China). Osteosarcoma cell lines and HEK-293 were cultured in DMEM supplemented with 10% FBS (Gibco, Gran Island, NY, USA), 100 U/mL penicillin, and 100 U/mL streptomycin (Invitrogen, Carlsbad, CA, USA), while hFOB1.19 cells were maintained in Ham’s F12/ DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. All the cells were incubated at 37°C with 5% CO2.
According to the information of hsa_circ_0007031 (circTUBGCP3) from GenBank, the sequences of the short siRNAs (GCAATAATGTGGTCTACAA) targeting spliced junction of circTUBGCP3 and negative control-siRNA (si-NC) were constructed by RiboBio (Guangzhou, China). The synthetic siRNA sequences were subcloned into the pcDNA3.1 vector (Invitrogen). hsa_circ_0007031 down-regulation was achieved through pcDNA3.1-siRNA transfection using Lipofectamine 3000 reagent according to the manufacturer’s instructions. The surviving cells were continuously cultured as stable mass transfectants.
According to the information of hsa_circ_0007031 (circTUBGCP3) from GenBank, the sequences of the short siRNAs (GCAATAATGTGGTCTACAA) targeting spliced junction of circTUBGCP3 and negative control-siRNA (si-NC) were constructed by RiboBio (Guangzhou, China). The synthetic siRNA sequences were subcloned into the pcDNA3.1 vector (Invitrogen). hsa_circ_0007031 down-regulation was achieved through pcDNA3.1-siRNA transfection using Lipofectamine 3000 reagent according to the manufacturer’s instructions. The surviving cells were continuously cultured as stable mass transfectants.
7
Culturing Human Osteosarcoma and Osteoblast Cell Lines
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The human osteosarcoma cell lines 143B and U2OS and human osteoblast cell line hFOB1.19 were obtained from the China Centre for Type Culture Collection (Wuhan, China). All 143B and U2OS cells were cultured in RPMI-1640 (Invitrogen, Carlsbad, CA, USA) medium supplemented with 10 % fetal bovine serum (FBS; Tian Hang), and they were propagated in a humidified environment with 5 % CO2 at 37 °C. hFOB1.19 was cultured in DMEM/F12 (Invitrogen) medium supplemented with 10 % fetal bovine serum (FBS; Tian hang) and geneticin (400 μg/ml, Sigma) and was incubated in an incubator with 5 % CO2 at 37 °C.
8
Human Osteoblast Cell Cultivation
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Cell Revival and Subculture Human foetal osteoblast cell line hFOB 1.19 (CRL-11372) was purchased from ATCC (American Type Cell Culture) (Manassas, VA 20110, USA). The hFOB 1.19 cells at passage 16 was cultured in Dulbecco's Modified Eagle Medium F-12 nutrient mixture (DMEM/F12) (Invitrogen GmBH, Karlsruhe, Germany), which was supplemented with 10% foetal bovine serum (FBS) (Invitrogen GmBH, Karlsruhe, Germany) and 1% penicillin/streptomycin serum (Invitrogen GmBH, Karlsruhe, Germany) (standard medium). The cells were incubated in 5% CO 2 37 °C humidified incubator (Sheldon, US) and monitored closely for 24 h. All procedures were carried out in an aseptic condition.
9
Osteosarcoma Cell Culture Protocol
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All OS cells, including Saos-2, MG63, HOS, 143B and U2OS, and hFOB1.19 were purchased from the ATCC (American Type Culture Collection) (Manassas, United States). OS cells were cultured in DMEM/F12 medium (Life Technologies, NY) with 10% fetal bovine serum (Gibco, NY) and streptomycin/penicillin (1%, Gibco, CA) and incubated with 5% CO2 at 37°C until 80–85% confluence. The hFOB 1.19 cells were cultured in DMEM/F-12 (Life Technologies, NY) containing 10% fetal bovine serum (Gibco, NY), streptomycin/penicillin (1%, Gibco, CA) and 0.3 mg/mL of G418 and incubated with 5% CO2 at 33.5°C.
10
Culturing Human Osteosarcoma Cell Lines
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All human OS cells, including Saos-2, U-2 OS, MG-63, MNNG, 143 B, and the normal osteoblast cell line hFOB 1.19, were obtained from the American Type Culture Collection (ATCC, Manassas, US). OS cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, US) supplemented with 10% fetal bovine serum (FBS) (Gibco, NY) and 1% penicillin/streptomycin (PS; Gibco, CA). The hFOB 1.19 cells were cultured in Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (DMEM/F12) (Life Technologies, NY) with 0.3 mgl/mL of G418, 10% FBS and 1% PS. All cells were incubated with 5% CO2 at 37°C.
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