The cell line GES-1 cells (ATCC) and BGC823 cells (ATCC) were preserved in this laboratory. The cells were placed in DMEM medium containing 10% fetal bovine serum, 1 × 105 IU/L penicillin, and 1 × 105 IU/L streptomycin conventional culture in an incubator at 37°C and 5% CO2.
Bgc 823 cells
Manufactured by American Type Culture Collection
Sourced in United States
BGC-823 cells are a human gastric carcinoma cell line. The cells are adherent and derived from a primary gastric adenocarcinoma.
Automatically generated - may contain errors
Lab products found in correlation
Ges 1 cells, by American Type Culture Collection (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Ly294002, by Merck Group (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Abcg2, by Cell Signaling Technology (1 mentions)
Ow cytometer, by BD (1 mentions)
Lipofectamine rnaimax kit, by Thermo Fisher Scientific (1 mentions)
Hek293t cell line, by American Type Culture Collection (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Dubelcco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
9 protocols using bgc 823 cells
1
Culturing GES-1 and BGC823 Cell Lines
2
Gastric Cancer Treatment with JPYW
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Human gastric cancer BGC823 cells were purchased from ATCC. Nude mice were purchased from Berke Biology co. LTD. China. JPYW was formed by Astragalus propinquus 15 g, Codonopsis pilosula 15 g, Atractylodes macrocephala 10 g, Angelica sinensis 10 g, Paeonia lactiflora 10 g, the pericarp of Citrus reticulata 6 g, ginger processed Pinellia ternata 10 g, Sparganium stoloniferum 10 g, Curcuma zedoaria 10 g, Salvia chinensis 30 g, Hedyotis diffusa 30 g, and Liquiritia 5 g. The abovementioned traditional Chinese medicinal materials were used to prepare a 400-ml decoction by the pharmaceutical department of Jiangsu Provincial Hospital of TCM. After filtraltion and concentration the decoction, we obtained JPYW stock with a concentration of 2.0 g/mL. Culture medium was used to dilute the stock to relevant concentration for experiments in vitro, while PBS was used to dilute the stock to relevant concentration for experiments in vivo. MRP1, MDR1, ABCG2, AKT, p-AKT, caspase-3 and bcl-2 antibodies were purchased from Cell Signaling Technology, Inc. USA. LY294002 was purchased from Sigma Aldrich Co. USA. All other reagents used were of analytical grade.
3
Cellular Uptake of Rhodamine-B Labeled PPN-S21i Nanoparticles
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The cellular uptake analysis was performed by confocal laser scanning microscopy (CLSM) and flow cytometer. BGC-823 cells (ATCC, USA) were cultured in RPMI-1640 media supplemented with 10% FBS. 2×10 5 cells were seeded in each well of 12-well plate and kept aside for 24h. The cells were treated with fresh medium containing the PPN-S21i nanoparticle containing rhodamine-B as a fluorescent tracker. The nanoparticles were incubated for 3h and then washed twice with PBS and then stained with Lysotracker Green for 10 min. The cells were again washed and fixed with 4% paraformaldehyde. The cells were observed under Leica Microsystems, Mannheim, Germany. The cellular uptake was further studied by flow cytometer (BD Biosciences, San Diego, CA). The cells were treated in the same manner as mentioned above and then collected by scrapping and 10,000 events were recorded.
4
Cell Viability Assay of Nanoparticles
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The cell viability assay was performed by cell counter kit-8 (CCK8) assay. To begin this assay, 8×10 3 BGC-823 cells (ATCC, USA) were seeded in each well of 96 well plates and incubated for 24h in 100 µl volume. The cells were then treated with a SMV, SMV+miR-21i and PPN-S20i nanoparticles in a concentration range from 1-100 µg/ml. For all concentration of SMV, miR-21i was fixed at 50 µg/ml. The cells were incubated for 24h. The cells were washed carefully and 10 µl of CCK-8 solution was added to each well of 96 well-plate and incubated for 1h at 37°C. The respective absorbance was measured using a microplate reader at 460 nm using BioTek, USA.
5
Cell Viability Assay of Nanoparticle Treatments
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The cell viability assay was performed by cell counter kit-8 (CCK8) assay. To begin this assay, 8×10 3 BGC-823 cells (ATCC, USA) were seeded in each well of 96 well plates and incubated for 24h in 100 µl volume. The cells were then treated with a SMV, SMV+miR-21i and PPN-S20i nanoparticles in a concentration range from 1-100 µg/ mL. For all concentration of SMV, miR-21i was xed at 50 µg/ mL. The cells were incubated for 24h. The cells were washed carefully and 10 µl of CCK-8 solution was added to each well of 96 well-plate and incubated for 1h at 37°C. The respective absorbance was measured using a microplate reader at 460 nm using BioTek, USA.
6
Cellular Uptake Analysis by CLSM and Flow Cytometry
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The cellular uptake analysis was performed by confocal laser scanning microscopy (CLSM) and ow cytometer. BGC-823 cells (ATCC, USA) were cultured in RPMI-1640 media supplemented with 10% FBS. 2×10 5 cells were seeded in each well of 12-well plate and kept aside for 24h. The cells were treated with fresh medium containing the PPN-S21i nanoparticle containing rhodamine-B as a uorescent tracker. The nanoparticles were incubated for 3h and then washed twice with PBS and then stained with Lysotracker Green for 10 min. The cells were again washed and xed with 4% paraformaldehyde. The cells were observed under Leica Microsystems, Mannheim, Germany. The cellular uptake was further studied by ow cytometer (BD Biosciences, San Diego, CA). The cells were treated in the same manner as mentioned above and then collected by scrapping and 10,000 events were recorded.
7
Modulating microRNA-29c-3p and Collagens in Gastric Cancer
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Human gastric adenocarcinoma BGC-823 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum and penicillin-streptomycin. Cells were maintained at 37°C in a 5% CO2 Atmosphere. To upregulate the expression of microRNA-29c-3p in BGC-823 cells, microRNA-29c-3p mimics, or a negative control sequence, were transfected into cells using the Lipofectamine® RNAimax kit (Thermo Fisher Scientific, MA, USA). To knock-down the expression of COL1A1 and COL4A1 in BGC-823 specific siRNA sequneces (COL1A1-siRNA and COL4A1-siRNA) were transfected into cells using Lipofectamine® RNAimax kit. To upregulate the expression of COL1A1 and COL4A1 expression vectors were constructed based on pcDNA3.1 and transfected into cells using Lipofectamine® 2000 Kit. Empty plasmids served as negative control.
8
Cell Line Culturing and shRNA Transfection
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HeLa cells, BGC823 cells, and HEK293T cell lines from American Type Culture Collection were cultured in Dubelcco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA, 12800-017) supplemented with 10% fetal bovine serum and maintained at 37 °C in a humidified incubator with 5% CO2. HeLa cells stably expressing GFP-LC3 were kindly provided by Dr. Li Yu (Tsinghua University).
The shRNA targeting the RNF115 gene was synthesized by GenePharma (Suzhou, China) and the targeting sequence was CGTCTGAATAGAATTAATT. The negative control shRNA sequence consisted of an oligonucleotide sequence with no sequence homology to any known human gene. NEOFECTTM DNA transfection reagent was used for transient transfection of plasmids, according to manufacturer’s instructions. For shRNA transfection, cells were transfected with Lipofectamine3000 according to manufacturer’s instructions (Invitrogen, L3000015).
The shRNA targeting the RNF115 gene was synthesized by GenePharma (Suzhou, China) and the targeting sequence was CGTCTGAATAGAATTAATT. The negative control shRNA sequence consisted of an oligonucleotide sequence with no sequence homology to any known human gene. NEOFECTTM DNA transfection reagent was used for transient transfection of plasmids, according to manufacturer’s instructions. For shRNA transfection, cells were transfected with Lipofectamine3000 according to manufacturer’s instructions (Invitrogen, L3000015).
9
Culturing Human Gastric Cancer Cells
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Human gastric adenocarcinoma BGC-823 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI-1640 culture medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids, and antibiotics. Cell cultures were maintained in a 5% CO2 incubator with controlled temperature and humidity.
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