Four human OS cell lines, MNNG (Catalogue No.: HTX1631), HOS (Catalogue No.: HTX3087), U2OS (Catalogue No.: HTX1634), and 143B (Catalogue No.: HTX2774), were provided by Otwo Biotech (ShenZhen) Inc. (Shenzhen, China). And one osteoblast cell line named hFOB1.19 (Catalogue No.: CL-0353) that was used as standard control was provided by another company, Procell Life Science&Technology Co., Ltd. (Wuhan, China). All the OS cell lines were incubated with a complete growth medium consisting of Dulbecco's modified Eagle's medium (DMEM, Gibco, Shanghai, China), 10% of fetal bovine serum (FBS, Tianhang, Zhejiang, China), and 1% of dual antibiotics (penicillin and streptomycin) (Solarbio, Beijing, China) in an incubator with the atmosphere condition of humidity and the concentration of carbon dioxide (CO2) at 5%; the temperature was set 37°C. At the same time, hFOB1.19 cells were cultured with a particular culture medium for human osteoblast (CM-H111, Procell Life Science & Technology Co., Ltd.) in a cell culture equipment with the atmosphere condition of humidity and the concentration of carbon dioxide (CO2) at 5%; the temperature was set at 34°C. And they were passaged with the ratio of 1 : 3 after the cells reached 90% of confluence.
Hfob1
Manufactured by Procell
Sourced in China
The HFOB1.19 is a laboratory instrument designed for performing high-frequency oscillation analysis. It is a versatile tool used in various scientific and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Saos 2, by Procell (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Cm h111, by Procell (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by ExCell Bio (1 mentions)
Si nc, by GenePharma (1 mentions)
Penicillin streptomycin, by Procell (1 mentions)
Sh circ, by GenePharma (1 mentions)
Fetal bovine serum (fbs), by Procell (1 mentions)
Si col1a1, by GenePharma (1 mentions)
Sw1353, by Procell (1 mentions)
Dulbecco modified eagle medium (dmem), by BD (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Transwell chamber, by Corning (1 mentions)
Mccoy s 5a, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg, by OriGene (1 mentions)
Fibronectin rabbit polyclonal antibody, by Abcam (1 mentions)
Ab32572, by Abcam (1 mentions)
Ab32072, by Abcam (1 mentions)
16 protocols using hfob1
1
Culturing and Characterizing Osteosarcoma Cell Lines
2
Culturing Human Osteoblast and Osteosarcoma Cells
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Human osteoblast cell line (HfoB1.19) and human OS cell lines (MG63, U2OS, and Saos-2) were all purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China).
Cell culture: HfoB1.19 cells, MG63, U2OS, and Saos-2 OS cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS). The media were purchased from Procell Life Science & Technology Corporation (Wuhan, China), and the FBS were purchased from Hyclone (South Logan, UT, USA). The medium contained penicillin (100 U/mL) and streptomycin (100 U/mL). All cell lines were grown in a 37°C incubator with 5% CO2.
Cell culture: HfoB1.19 cells, MG63, U2OS, and Saos-2 OS cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS). The media were purchased from Procell Life Science & Technology Corporation (Wuhan, China), and the FBS were purchased from Hyclone (South Logan, UT, USA). The medium contained penicillin (100 U/mL) and streptomycin (100 U/mL). All cell lines were grown in a 37°C incubator with 5% CO2.
3
Osteosarcoma Cell Line Cultivation Protocol
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Human OS cell lines (U2OS, HOS, Saos2 and MG63) were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China). OS cells were grown in DMEM (Invitrogen, Waltham, MA, USA) supplemented with 10% FBS (ExCell Bio, Shanghai, China) and 1% penicillin-streptomycin (HyClone, Logan, UT, USA). These cells were cultured in a humidified incubator with containing 5% CO2 (link) at 37°C. Human immortalized osteoblasts (hFOB 1.19) were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China), and cultured in DMEM/F12 (Invitrogen) containing 0.3 mg/mL G418 (TargetMol, Boston, MA, USA) at 34°C. The cells were routinely checked for mycoplasma contamination.
4
Osteoblast and Osteosarcoma Cell Culture
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Normal osteoblasts (hFOB1.19) and osteosarcoma cells (HOS, Saos-2, and SW1353) were obtained from ProCell (China). SW1353 (Leibovitz’s L-15 Medium; Procell), Saos-2 (McCoy’s 5A; Procell), HOS (MEM; Procell), and hFOB1.19 (DMEM/F12; Procell) were maintained in media containing 1% penicillin-streptomycin (Procell) and 10% fetal bovine serum (FBS, Procell). Culture was performed under a 5% CO2 atmosphere at 37°C.
Small interfering RNAs (siRNAs) targeting circ_0020378 (si-circ_0020378), COL1A1 (si-COL1A1), miR-339-3p inhibitor and mimic, and their respective negative controls (si-NC, inhibitor NC, and mimic NC) were purchased from GenePharma, China. Recombinant lentiviruses (multiplicity of infection [MOI] = 50) carrying short hairpin RNAs (shRNAs) targeting either circ_0020378 (sh-circ) or sh-NC were purchased from GenePharma. Lipo3000 was used to introduce synthetic nucleotides (siRNAs, mimics, and inhibitors) into Saos-2 and HOS cells. After a 48-h transfection period, the efficacy of transfection was verified by RT-qPCR. For shRNA lentivirus infection, approximately 80% confluent Saos-2 cells were incubated with the virus particles (MOI = 50). After 14 days, the survivors were screened by adding 0.5 μg/mL puromycin. Positive monoclonal cells were expanded and verified using RT-qPCR.
Small interfering RNAs (siRNAs) targeting circ_0020378 (si-circ_0020378), COL1A1 (si-COL1A1), miR-339-3p inhibitor and mimic, and their respective negative controls (si-NC, inhibitor NC, and mimic NC) were purchased from GenePharma, China. Recombinant lentiviruses (multiplicity of infection [MOI] = 50) carrying short hairpin RNAs (shRNAs) targeting either circ_0020378 (sh-circ) or sh-NC were purchased from GenePharma. Lipo3000 was used to introduce synthetic nucleotides (siRNAs, mimics, and inhibitors) into Saos-2 and HOS cells. After a 48-h transfection period, the efficacy of transfection was verified by RT-qPCR. For shRNA lentivirus infection, approximately 80% confluent Saos-2 cells were incubated with the virus particles (MOI = 50). After 14 days, the survivors were screened by adding 0.5 μg/mL puromycin. Positive monoclonal cells were expanded and verified using RT-qPCR.
5
Cell Culture Optimization for Bone Research
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143B, U2OS, MG63, and Saos-2 cells were provided by Bena Culture Collection Biotechnology Co., Ltd., (Beijing, China). hFOB1.19 cells were obtained from Procell (Wuhan, China). Saos-2, MG63, hFOB1.19 and U2OS cells were cultivated within the Dulbecco’s Modified Eagle Medium (DMEM, BD, USA) containing fetal bovine serum (FBS, 10%, Hyclone, Logan, UT, USA). The RMPI1640 medium containing 10% FBS was used to the 143B cells. All cells were cultivated at 37°C in an incubator under CO2 atmosphere.
6
Silencing PTN in Osteoblast and OSA Cells
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The osteoblast cell line (hFOB1.19) and human OSA cell lines (HOS, Saos-2 and 143B) were purchased from the Procell Life Science&Technology Co.,Ltd. (Wuhan, China). The cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and were grown in an incubator at 37°C with 5% CO2. The silencing RNA against PTN (si-PTN) was synthesized and purchased from TsingkeBiotechnology Co.,Ltd. (Beijing, China). The sequence of si-PTN is shown in Supplementary Table 1
7
Osteosarcoma Tissue and Cell Line Analysis
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This study was approved by the ethics committee of the General Hospital of Ningxia Medical University (No. KYLL-2021-562), and a written informed consent form was signed by all patients. Ten osteosarcoma samples and matched adjacent normal tissues were collected from August 2021 to August 2022, and postoperative pathology indicated osteosarcoma. The specimens were frozen and stored at -80°C until use.
Osteosarcoma cell lines (U-2OS, MG-63, and Saos-2) and a normal osteoblast cell line (hFOB 1.19) were purchased from Procell Life Science & Technology (Wuhan, Hubei, China), and verified using STR genotyping. U-2OS and Saos-2 cells were cultured in McCoy's 5A medium supplemented with 10% FBS (fetal bovine serum) and 1% penicillin and streptomycin. MG-63 cells were grown in minimum essential medium supplemented with 10% FBS and 1% penicillin and streptomycin. hFOB 1.19 cells were cultured in F12 medium and Dulbecco’s modified Eagle medium supplemented with 0.3 mg/ml G418, 10% FBS, and 1% penicillin and streptomycin. hFOB 1.19 cells were maintained in an incubator at 34°C with 5% CO2, and all other cells were maintained at 37°C with 5% CO2.
Osteosarcoma cell lines (U-2OS, MG-63, and Saos-2) and a normal osteoblast cell line (hFOB 1.19) were purchased from Procell Life Science & Technology (Wuhan, Hubei, China), and verified using STR genotyping. U-2OS and Saos-2 cells were cultured in McCoy's 5A medium supplemented with 10% FBS (fetal bovine serum) and 1% penicillin and streptomycin. MG-63 cells were grown in minimum essential medium supplemented with 10% FBS and 1% penicillin and streptomycin. hFOB 1.19 cells were cultured in F12 medium and Dulbecco’s modified Eagle medium supplemented with 0.3 mg/ml G418, 10% FBS, and 1% penicillin and streptomycin. hFOB 1.19 cells were maintained in an incubator at 34°C with 5% CO2, and all other cells were maintained at 37°C with 5% CO2.
8
Investigating Osteosarcoma Cell Signaling
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Osteosarcoma cell lines (SAOS2, MG63 and U2OS) and the human osteoblast cell line (hFOB1.19) were purchased from Procell Co. Ltd (Wuhan, China). PRR11 rabbit anti-human polyclonal antibody (Ab237526), β-catenin rabbit anti-human monoclonal antibody (ab32572), p-β-catenin rabbit anti-human monoclonal antibody (ab75777), GSK-3β mouse anti-human monoclonal antibody (ab93926), p-GSK-3β rabbit anti-human monoclonal antibody (ab68476), c-Myc rabbit anti-human monoclonal antibody (ab32072), CyclinD1 rabbit anti-human monoclonal antibody (ab16663), E-cadherin mouse anti-human monoclonal antibody (Ab1416), Vimentin rabbit anti-human monoclonal antibody (ab92547), Fibronectin rabbit polyclonal antibody (ab2413), total RNA extraction kit were purchased from Abcam (USA). McCoy's 5A and 10% fetal bovine serum-containing DEME were purchased from GIBCO (Thermo Fisher Scientific, USA). Goat anti-mouse IgG, goat anti-rabbit IgG, DAB color kit was purchased from OriGene (USA). The reverse transcription kit HiScript reverse transcriptase and SYBR Green Master Mix were purchased from Novozan Biotechnology Co., Ltd (Nanjing, China). Transwell chamber were purchased from Corning, (USA), and CCK8 kit was purchased from MCE, (USA). PRR11 targeting SiRNA was purchased from Jima (Shanghai, China).
9
Osteosarcoma and Osteoblast Cell Culture
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Human osteosarcoma cells (Saos2, 143B) and normal osteoblast cell line (hFOB 1.19) were provided by the Procell Company (Wuhan, China). Saos2 cells were cultured in McCoy’s medium supplemented with 15% FBS, while 143B cells were in RPMI 1640 medium supplemented with 10% FBS. hFOB cells were cultured in DMEM/F-12 medium. All osteosarcoma cell lines were maintained in a humidified incubator at 37 °C with 5% CO2, while hFOB 1.19 cells were cultured at 35 °C with 5% CO2.
10
Osteosarcoma Cell Lines Overexpressing TNFRSF21
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Human osteoblast cell line hFOB1.19 (RRID: CVCL_3708) and three human osteosarcoma cell lines HOS (RRID: CVCL_0312), MG63 (RRID: CVCL_0426) and U2OS (RRID: CVCL_0042) were used in this study. Osteosarcoma cell lines were purchased from Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Shanghai, China), hFOB1.19 was purchased from Procell Biotechnology Co., Ltd. (Wuhan, China). Before experiments, DNA types of cell lines were identified by short tandem repeat (STR) profiling and compared with DSMZ data. All experiments were performed with mycoplasma-free cells which were cultured according to the instructions.
Lentivirus containing pLVX-TNFRSF21-Puro and pLVX-Puro (vector) were purchased from Genechem (Shanghai, China) and used to infect HOS and MG63 according to the manufacturer’s protocol. Stably transfected cells were selected with puromycin and validated by polymerase chain reaction (PCR) and western blot.
Lentivirus containing pLVX-TNFRSF21-Puro and pLVX-Puro (vector) were purchased from Genechem (Shanghai, China) and used to infect HOS and MG63 according to the manufacturer’s protocol. Stably transfected cells were selected with puromycin and validated by polymerase chain reaction (PCR) and western blot.
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