The HEK293T, 786-O and 769P cell lines were purchased from the American Type Culture Collection (ATCC). The HEK293T, 786-O and 769-P cells were respectively cultured in RPM1-DMEM medium (with L-glutamine) and RPM1-1640 medium (with L-glutamine) (Fcmacs Biotech, Nanjing, China) in a 5% CO2 incubator at 37° C. Both kinds of medium were complete medium supplemented with 10% fetal bovine serum (FBS) (Fcmacs Biotech) and 1% penicillin and streptomycin (Sangon, Shanghai, China).
Penicillin and streptomycin
Manufactured by Sangon
Sourced in China, United States
Penicillin and streptomycin are antibiotics commonly used in laboratory settings. Penicillin is a beta-lactam antibiotic that inhibits the synthesis of bacterial cell walls, while streptomycin is an aminoglycoside antibiotic that disrupts bacterial protein synthesis. These antibiotics are used to prevent or treat bacterial contamination in laboratory cultures and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Penicillin and streptomycin, by Cytiva (1 mentions)
Lv nc shrna, by GenePharma (1 mentions)
Hcclm 3, by GenePharma (1 mentions)
Hepg2, by GenePharma (1 mentions)
Fetal bovine serum (fbs), by Cytiva (1 mentions)
Puromycin, by GenePharma (1 mentions)
Dulbecco modified eagle medium (dmem), by Cytiva (1 mentions)
Hepg2, by Procell (1 mentions)
Mhcc97h, by Shanghai Zhong Qiao Xin Zhou Biotechnology (1 mentions)
Hcclm3, by Shanghai Zhong Qiao Xin Zhou Biotechnology (1 mentions)
Hepg2, by Cytiva (1 mentions)
Antibiotic mixture, by Sangon (1 mentions)
L glutamine, by Sangon (1 mentions)
Sk rc 39 cell line, by Cellcook (1 mentions)
Rpm1 dmem, by Fcmacs (1 mentions)
Rpm1 1640, by Fcmacs (1 mentions)
Fetal bovine serum (fbs), by Fcmacs (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
6 protocols using penicillin and streptomycin
1
Cell Culture Conditions for HEK293T, 786-O and 769-P
2
Liver Cancer Cell Line Cultivation and Manipulation
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The human LIHC cell lines HepG-2 and Huh-7 were obtained from Procell Life Science&Technology Co., Ltd. (Wuhan, China). The human LIHC cell lines MHCC97-H and HCCLM-3 were obtained from Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Shanghai, China). The human normal liver cell line HL-7702 (L02) were obtained from Guan&Dao Biological Engineering Co., Ltd. (Shanghai, China). HepG-2, Huh-7, MHCC97-H and HCCLM-3 cells were cultured in DMEM (Cytiva, Utah, USA) with 10% FBS (Gibco, NY, USA) plus 1% penicillin and streptomycin (Sangon, Shanghai, China). L02 cell line was cultured in RIPA 1640 medium (Cytiva, Utah, USA) with 10% FBS plus 1% penicillin and streptomycin. All of the cells were cultured under humidified condition with 5% CO2 at 37 °C. The authentication of these cell lines was performed via comparisons with the STR database.
The lentivirus vectors LV–NC–shRNA, LV-CCDC115-shRNA and LV-CCDC115-OE were purchased from GenePharma (Shanghai, China). HepG-2 and HCCLM-3 cells were infected with lentivirus (MOI: HepG-2 cell = 80 pfu/cell; HCCLM-3 cell = 100 pfu/cell) with hexadimethrine bromide (5 μg/mL, GenePharma) for 72 h and puromycin (GenePharma) at the concentration of 2 μg/mL (HepG-2) or 2.5 μg/mL (HCCLM-3) was used to select for stably transfected cells.
The lentivirus vectors LV–NC–shRNA, LV-CCDC115-shRNA and LV-CCDC115-OE were purchased from GenePharma (Shanghai, China). HepG-2 and HCCLM-3 cells were infected with lentivirus (MOI: HepG-2 cell = 80 pfu/cell; HCCLM-3 cell = 100 pfu/cell) with hexadimethrine bromide (5 μg/mL, GenePharma) for 72 h and puromycin (GenePharma) at the concentration of 2 μg/mL (HepG-2) or 2.5 μg/mL (HCCLM-3) was used to select for stably transfected cells.
3
Isolation and Culture of Murine Adipose Stromal Cells
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Epididymal fat pads were obtained from C57BL/6 or T2D mice and washed with phosphate-buffered saline (PBS). Tissues were digested with 0.1% collagenase type І (Sigma-Aldrich, St. Louis, MO, USA) by incubation in a shaker at 37°C for 15 to 20 minutes. The digestion was terminated by a culture medium containing 10% fetal bovine serum (FBS, Gibco; Langley, OK, USA). The mixture was then centrifuged for 5 min at 1,200 rpm. After centrifugation, the cell pellet was resuspended in complete medium made of Dulbecco's modified Eagle's medium/F12 medium supplemented with 10% FBS and 1% penicillin and streptomycin (Sangon Biotech Co., Ltd., Shanghai, China) [10 (link)]. Cells were cultured in 37°C at 5% CO2 atmosphere and were passaged at 90% confluence with 0.25% Trypsin-EDTA. The third passage ASCs were used in subsequent experiments.
4
Culturing SK-RC-39 and HK-2 Cell Lines
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Human pRCC SK-RC-39 cell line and normal renal cell line HK-2 were purchased from Cellcook Biotech (Guangzhou, China). The cell mediums (including RPM1-DMEM and RPM1-1640) and Fetal Bovine Serum (FBS) were purchased from Fcmacs Biotech (Nanjing, China). Antibiotic mixture (penicillin and streptomycin) and l -glutamine were purchased from Sangon Biotech (Shanghai, China). 57 mL FBS and 5.7 mL Antibiotic mixture as well as 5.7 mL l -glutamine were added into cell mediums to generate complete mediums. SK-RC-39 and HK-2 were fed in complete 1640 and DMEM medium, respectively. Both the two kinds of cell lines were cultured in the 5% CO2 incubator at 37 °C.
5
Cultivation of THP-1 Monocytic Leukemia Cells
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Human monocytic leukemia THP-1 cells (purchased from Shanghai Institutes for Biological Sciences) were maintained in RPMI-1640 medium (Gibco, MD, USA), which was supplemented with 10% fetal bovine serum (FBS, Gibco, MD, USA) and 100 U of penicillin and streptomycin (Sangon Biotech, Shanghai, China) in a 5% CO2-humidified incubator at 37 °C.
6
Functional Analysis of HIF Isoforms
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For functional analysis, open reading frame (ORF) DNA of hifαs was cloned into a pCS2 + vector to obtain pCS2-hifαs constructs. Primers for this part were shown in Additional le 1. HEK293T cells were grown in medium M199 (Gibco, USA) supplemented with 10% FBS (Gibco, Australia) and 0.5% penicillin and streptomycin (Sangon, China), in a constant temperature cell incubator at 37 ℃ under normal oxygen (about 5% CO 2 ). Cells were plated onto 24 well plates (1-2×10 5 cells/well) 24 hours to 55% con uency prior to transfection. The plasmids were transiently transfected into HEK293T cells using Lipofectamine™ 2000
Transfection Reagent (Invitrogen, USA). The cells were then incubated for 48 hours to allow DNA uptake and genes expression. Each experiment was repeated three times.
Luciferase reporter assay HEK293T cells were cotransfected with pCS2-hifαs (pCS2-hif1αa/b, pCS2-hif2αa/b, pCS2-hif3αa/b) or pCS2 + plasmid with the pGL3-HRE. pGL3-HRE is a HRE reporter gene that was constructed by reference to Huang et al. (2017) (link). To control for transfection e ciency, pRL-TK, a Renilla luciferase reporter plasmid, was cotransfected. Luciferase reporter assay was carried out using Dual-Luciferase Reporter Assay System (Promega, USA). Each experiment was repeated in triplicate.
Transfection Reagent (Invitrogen, USA). The cells were then incubated for 48 hours to allow DNA uptake and genes expression. Each experiment was repeated three times.
Luciferase reporter assay HEK293T cells were cotransfected with pCS2-hifαs (pCS2-hif1αa/b, pCS2-hif2αa/b, pCS2-hif3αa/b) or pCS2 + plasmid with the pGL3-HRE. pGL3-HRE is a HRE reporter gene that was constructed by reference to Huang et al. (2017) (link). To control for transfection e ciency, pRL-TK, a Renilla luciferase reporter plasmid, was cotransfected. Luciferase reporter assay was carried out using Dual-Luciferase Reporter Assay System (Promega, USA). Each experiment was repeated in triplicate.
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