Primary BMMSCs were isolated as previously described (Liao et al., 2016 ). Briefly, BMMSCs from 3‐month‐old mice were obtained and cultured in α‐MEM (Gibco BRL) supplemented with 20% fetal bovine serum (FBS; Thermo Electron), 0.292 mg/ml glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (Invitrogen). Cells were then plated in the 10 cm dish (Costar) at 37°C in 5% CO2, and the medium was changed every 3 days. BMMSCs at passage 1 were used in the experiments. Bay K8644 (Abcam) was dissolved in DMSO (0.1% culture concentration).
10 cm dish
Manufactured by Corning
Sourced in United States
The 10 cm dish is a laboratory equipment item used for various applications. It provides a flat, circular surface area of 10 centimeters in diameter, which can be used for a range of purposes such as cell culture, sample preparation, or general containment tasks in a controlled laboratory environment.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Glutathione assay kit, by Merck Group (2 mentions)
Dulbeccco s modified eagle s media d mem, by Thermo Fisher Scientific (2 mentions)
Tib 202, by American Type Culture Collection (2 mentions)
Nutrient mixture f 12, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Matrigel, by Corning (2 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Bay k 8644, by Abcam (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Megascripttm t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
Gene pulser, by Bio-Rad (1 mentions)
Penicillin g streptomycin sulfate, by Thermo Fisher Scientific (1 mentions)
D5796, by Merck Group (1 mentions)
Celltracker green cmfda, by Thermo Fisher Scientific (1 mentions)
22 protocols using 10 cm dish
1
Isolation and Culture of BMMSCs
2
Efficient Lentiviral Knockdown of APE1 in 293T Cells
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The lentiviral vector PDS019-pL-shRNA-F (NovoBio Scientific, Shanghai, China) was applied to synthesize APE1 shRNA-containing plasmid (PDS019_pL_shRNA_GFP-hAPE1), as described in a previously published study (17 (link)). We used 3 pairs of primers based on the APE1 gene sequence (NM_080649.2), and synthesized 3 lentiviral plasmids, including VL1275-PDS019_pL_shRNA_GFP-hAPE1-446, VL1276-PDS019_pL_shRNA_GFP-hAPE1-609, and VL1277-PDS019_pL_shRNA_GFP-hAPE1-839 (APE1shRNA) (Figure 1A ). By examining interference efficiency, we found that APE1shRNA demonstrated the highest interference efficiency (81.82%) (Figure 1B ), as seen in the following experiments. Briefly, 1 day before the transfection of APE1shRNA, 293T cells (Shanghai Institute of Cells, Chinese Academy of Science, Shanghai, China) were seeded onto a 10-cm dish (Corning-Costar, Corning, NY, USA). The APE1shRNA plasmid and packing plasmids were transfected into 293T cells, according to the manufacturer’s instructions. Appropriately 72 h post-transfection, the supernatants of 293T cells were harvested by centrifuging at 2,000 ×g for 5 min at 4 °C. Cell supernatants were purified with a 0.5-µm syringe filter, and products were re-centrifuged at 20,000 ×g at 4 °C for 1 h. The APE1shRNA were packaged into lentiviral.
3
RNA Delivery via Electroporation in Huh 7.5.1 Cells
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The Jc1 and Jc1-S282T plasmids were linearised by Xba I digestion. The linearised plasmids DNA were transcribed by MEGAscriptTM T7 Transcription Kit (Invitrogen) after purification. RNAs were delivered to cells by electroporation as described previously [34] (link). Briefly, Huh 7.5.1 cells were washed twice and resuspended in serum-free Opti-MEM (Invitrogen) at 1 × 107 cells per ml. 10 μg RNA mixed with 400 μL cells in a 4-mm cuvette, then pulsed by Bio-Rad Gene Pulser at 0.27 kV, 100 Ω and 950 μF. The pulsed cells were plated in a 10 cm dish (Corning).
4
HeLa Cell Culturing and Preparation
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HeLa cells were cultured in a 10 cm dish (Corning) filled with Dulbecco's Modified Eagle's Medium (DMEM) at 37 °C in the presence of 5% CO2. The cells were cultured until 80% confluent, and the cells were washed with phosphate buffered saline without Ca2+ and Mg2+ (PBS(−)) after removing DMEM. Then the HeLa cells were prepared with trypsin–EDTA treatment. After trypsin–EDTA was removed, DMEM was added to the dish, and the cell suspension was transferred to a centrifuge tube. DMEM was prepared as follows: Dulbecco's modified Eagle's medium (D5796, Sigma) was mixed with Fetal bovine serum (10 vol%) (10437-028, Gibco) and Penicillin G–Streptomycin sulfate (5 vol%) (15070-063, Gibco). PBS(−) containing NaCl (8.00 g L−1), KCl (0.20 g L−1), Na2HPO4 (1.15 g L−1), and KH2PO4 (0.20 g L−1) was used for the experiments after autoclaving at 120 °C for 20 min.
5
Organoid Invasion Assay Protocol
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A total of 250,000 cells were resuspended in 1 mL of the corresponding media and 20 μL drops were placed in the lid of a 10 cm dish (Corning). The lid was flipped over the dish containing 5 mL of media in order to prevent evaporation. Hanging drops were incubated for 5 days at 37% C in a humidified atmosphere, during which formation of organoids was achieved. Before being included in 3D matrix for the invasion assay, the organoids were collected and labeled with 10 μM CellTracker™ Green CMFDA (Thermo Fisher, Waltham, USA) dye by incubating them in serum free media for 45 min at 5% CO2. Labeling solution was removed, and spheroids were washed in cell medium. To follow, spheroids were centrifuged at 300 rpm, immersed in 10 μL of phenol-red free Matrigel® (BD Biosciences) and placed in a 24 well-plate (Corning) The appropriate media containing G418 or puromycin was subsequently added to the well. Brightfield images were acquired at days zero and day two using an EVOS microscope (Advanced Microscopy Group, Life Technologies). Images were analyzed using Fiji ImageJ software and fold-change area was calculated using the following formula: Area (fold-change) = Area Day 2/Area Day 0.
6
Isolation and Culture of Murine BMSCs
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Murine BMSCs were obtained using a previous described protocol with minor modifications (Grassel et al., 2012 (link)). In brief, murine femur and humerus were dissected from Lrp5fl/fl or wild-type C57BL/6J mice at P14 after euthanasia and washed three times in cold phosphate buffered saline (PBS; Cytiva) with 1% penicillin-streptomycin (Sigma-Aldrich). The isolated long bones were then cut with sterile scissors, and the bone marrow was extracted by centrifuging at 500 g for 30 s. Pellet was resuspended in PBS and filtered with 70-μm cell strainer (BD Falcon). Cells were then resuspended and seeded in 10-cm dishes (Corning) in Dulbecco’s modified Eagle’s medium (DMEM) high glucose (Hyclone), 1% penicillin-streptomycin (Sigma-Aldrich), and 10% fetal bovine serum (FBS; Gibco). BMSCs were cultured at 37°C and 5% CO2, and the cell medium was changed every 48 h. After 80–90% cell confluence was reached, the adherent cells were washed twice in PBS, trypsinized using 0.25% Trypsin-EDTA (Thermo Fisher Scientific), centrifuged at 1,000 g for 3 min, and then seeded in 10-cm dish (Corning) at the next passage.
7
Culturing GBM8 Neurospheres for Cancer Research
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Low passage GBM8 cells have been cultured as neurospheres in Neurobasal medium (Gibco) supplemented with 3 mM GlutaMAX (Gibco), 1× B-27 supplement (an optimized serum-free proprietary supplement commercially available from Gibco), 0.5× N-2 supplement (a chemically defined, serum-free supplement from Gibco; components include transferrin, insulin, progesterone, putrescine and selenite), 0.5% Antibiotic-Antimycotic Solution (Corning), 20 ng/mL EGF (R&D Systems, MN, United States), and 20 ng/mL FGF (PeproTech, NJ, United States). Mature neurospheres were dissociated using NeuroCult Chemical Dissociation Kit (Mouse) (Stemcell Technologies, Canada). Approximately 0.5 × 106 cells were seeded in 10 mL fresh media in a 10 cm dish (Corning), and 1/3 volume of fresh medium was added every 3 days. Mature neurospheres were usually formed within 7∼10 days.
8
Gastric Cancer Cell Secretome Effect on Mesenchymal Stem Cells
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For gastric cancer cells, 5 × 105 of MFCs were planted in 10 cm dish (Corning, NY, USA) for 48 h. When the confluence of cells amounted to 70%, cell culture medium was harvested.
For mMSCs, 5 × 104 of mMSCs were seeded in each well of six‐well‐plate (Corning) and attached overnight. After treated with MFC‐CM or transfected with oligonucleotides for 48 h or pretreated with pyrrolidine dithiocarbamic acid (PDTC, Sigma‐Aldrich Shanghai Trading Co. Ltd, Shanghai, China) for 2 h before treated with MFC‐CM or transfection, cell culture medium was removed and refreshed for 24 h and then the cell culture supernatant was collected. All of the medium were then centrifuged at 1500 rpm for 10 min and filtered through a 0.22‐μm membrane (Merck Millipore, Darmstadt, Germany) and stored in −80°C until use.
For mMSCs, 5 × 104 of mMSCs were seeded in each well of six‐well‐plate (Corning) and attached overnight. After treated with MFC‐CM or transfected with oligonucleotides for 48 h or pretreated with pyrrolidine dithiocarbamic acid (PDTC, Sigma‐Aldrich Shanghai Trading Co. Ltd, Shanghai, China) for 2 h before treated with MFC‐CM or transfection, cell culture medium was removed and refreshed for 24 h and then the cell culture supernatant was collected. All of the medium were then centrifuged at 1500 rpm for 10 min and filtered through a 0.22‐μm membrane (Merck Millipore, Darmstadt, Germany) and stored in −80°C until use.
9
Cell Line Maintenance in RPMI
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Five cell lines (HEL, K562, U937, HL-60, and THP1) were maintained in RPMI 1640 medium (Wako, Japan) supplemented with 10% (v/v) fetal bovine serum (FBS), 50 U/mL penicillin, and 50 mg/mL streptomycin in a 10 cm dish (Corning, Inc., Corning, NY, USA). Cells were passaged twice per week.
10
Glutathione Quantification in Cells
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Cells were seeded in a 10-cm dish (Corning Inc., Corning, NY, USA) at a density of 1 × 106 cells and were cultured 24 h prior to drug treatment. The sample was deproteinized with 5 % 5-sulfosalicylic acid solution. The cellular level of glutathione was determined using a Glutathione Assay Kit (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer's protocol.
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