Cells were collected following transfection. A Transwell invasion chamber (Beyotime Institute of Biotechnology; polycarbonate pore membrane with pore size 8 µm) was placed in a 24-well cell culture plate. Matrigel (15 µg/ml; Beyotime Institute of Biotechnology) was placed on the surface of the filter membrane of the Transwell chamber, and following coagulation, RPMI 1640 serum-free medium (Beyotime Institute of Biotechnology) (37°C) was used for hydration for 30 min. 0.25%trypsin (Beyotime Institute of Biotechnology) was used for digestion of cells in the logarithmic growth phase. Following suspension and dilution of serum-free medium, 1×105 cells were inoculated in each chamber containing a volume of 200 µl, and 600 µl complete medium was added to the lower chamber. Following 24 h of incubation, the small chamber was removed. A cotton swab was used to wipe the non-invaded cells from the surface of the microporous membrane. The filter membrane was then fixed with methanol for 20 min. Crystal violet (Shanghai Qiaoxing Trading Corporation, Shanghai, China) staining was performed for 10 min, and under an optical microscope (magnification, x200), eight fields of view were randomly selected to perform the averaged cell count. The above experiment was repeated three times, with three wells per group.
Transwell chamber
Manufactured by Beyotime
Sourced in China
Transwell chambers are cell culture inserts designed to study cell migration, invasion, and permeability. They consist of a porous membrane that separates the upper and lower compartments of a culture well, allowing the exchange of media and solutes while physically separating the cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Crystal violet, by Beyotime (3 mentions)
Transwell chamber, by Corning (3 mentions)
Transwell chamber, by BD (2 mentions)
Matrigel, by Beyotime (1 mentions)
Trypsin, by Beyotime (1 mentions)
Matrigel, by BD (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Anti mtor, by Beyotime (1 mentions)
Anti β actin, by Beyotime (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Cck 8 kit, by Beyotime (1 mentions)
Histone 1, by Santa Cruz Biotechnology (1 mentions)
Cox 2, by Santa Cruz Biotechnology (1 mentions)
Cck 8 assay, by Beyotime (1 mentions)
Ripa assay, by Beyotime (1 mentions)
Matrigel, by Thermo Fisher Scientific (1 mentions)
Giemsa, by Beyotime (1 mentions)
8 protocols using transwell chamber
1
Transwell Invasion Assay for Cell Migration
2
Transwell Invasion Assay for Cancer Cells
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Migrated cells were detected by the transwell chamber (Corning Inc., Corning, NY, USA), and the transwell chamber was enveloped with matrigel (BD Biosciences) for invasion determination [23 (link)]. The upper chamber was added with 1 × 105 CaSki and SiHa cells, and then, 500 μL cell medium was pipetted into the lower chamber. The transwell chamber was incubated for 24 h; then cells from the upper chamber into the lower chamber were stained with 0.1% crystal violet (Beyotime). Cell pictures were saved at 100× magnification by an inverted microscope (Olympus), and cell number was counted under three fields of view.
3
Transwell Invasion Assay Protocol
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Cells from the different groups were suspended within the serum-free medium, followed by pancreatin digestion. The cell number of cells were counted and 200 μL of the cell suspension (including 5 × 103 cells) was added into the upper chamber, while 500μL DMEM supplemented with 10% FBS was added into the lower chamber of the transwell chamber (Corning, New York, USA). Thereafter, the cells were incubated a humidified incubator at 37°C for 24 hrs. The cells that have penetrated the transwell chamber were rinsed, followed by 4% formaldehyde fixation and staining with 0.1% crystal violet (Beyotime). The invading cells were observed with a microscope (Olympus Corporation, Japan) and counted. Each experiment was carried out in triplicate.
4
RPL22L1 Modulates Cell Proliferation
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The tissue microarray was purchased from Alenabio Co. Ltd. Foetal bovine serum (FBS) and Dulbecco's Modified Eagle's Medium (DMEM) were obtained from Thermo Fisher Inc. Anti‐RPL22L1 and the secondary antibodies were purchased from Proteintech. CCK‐8 kit, transwell chambers, enhanced chemiluminescence reaction kit (ECL), bovine serum albumin (BSA), LY294002, anti‐PI3K (p85), anti‐p‐PI3K (p85), anti‐Akt, anti‐p‐Akt (Ser473), anti‐mTOR, anti‐p‐mTOR (Ser2448) and anti‐β‐actin were obtained from Beyotime Biotechnology. RPL22L1 lentiviral activation particles and RPL22L1 shRNA lentiviral particles were purchased from Santa Cruz.
5
Molecular Assays for Cancer Research
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Cisplatin (cDDP), penicillin were from Sigma-Aldrich (USA). Antibodies against APE1, COX-2 and Histone 1 were from Santa Cruz Biotechnology (USA). Antibodies against β-tubulin, P-Akt, and Akt were from Abcam (USA). The CCK-8 assay, RIPA assay, Transwell® chambers and crystal violet were from Beyotime Corporation (China).
6
Measuring Cell Migration and Invasion
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Cell migration was determined by using a wound healing assay. Briefly, MDA-MB-231 cells were plated as monolayer at a density of 5×105 cells per well in a 6-well plate and grown to confluence, then the monolayer cells were scratched with a 10-μL micropipette tip and then replaced with fresh medium supplemented with 20 μM celecoxib. After incubation for 24 h, the wound distances were measured using NIS-Elements imaging software.
Cell invasion was conducted by using Transwell Chambers (Corning). The upper chamber was coated with 100 μL Matrigel (Invitrogen), and the lower chamber was filled with 500 μL DMEM supplemented with 10% FBS. MDA-MB-231 cells were plated at 3000 cells per chamber in the upper chamber containing 20 μM celecoxib. The Transwell Chambers were incubated at 37°C and 5% CO2 for 24 h. Cells on the upper surface of the insert were removed using a cotton swab, and cells that had migrated to the lower surface were stained with 2% crystal violet (Beyotime Biotechnology) for 30 min. Images of migrated cells were taken and the number of migrated cells was counted under a microscope in three randomly selected fields (magnification 100x).
Cell invasion was conducted by using Transwell Chambers (Corning). The upper chamber was coated with 100 μL Matrigel (Invitrogen), and the lower chamber was filled with 500 μL DMEM supplemented with 10% FBS. MDA-MB-231 cells were plated at 3000 cells per chamber in the upper chamber containing 20 μM celecoxib. The Transwell Chambers were incubated at 37°C and 5% CO2 for 24 h. Cells on the upper surface of the insert were removed using a cotton swab, and cells that had migrated to the lower surface were stained with 2% crystal violet (Beyotime Biotechnology) for 30 min. Images of migrated cells were taken and the number of migrated cells was counted under a microscope in three randomly selected fields (magnification 100x).
7
Sanguinarine Inhibits Cell Invasion
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Cell invasion assays were performed by the Transwell chambers (Beyotime) with polycarbonate membrane (8 μm). Polycarbonate membrane was pre‐coated with Matrigel (5 mg/ml) and rehydrated with FBS‐free DMEM medium (50 μl, 30 min., 37°C). Then, cells (5 × 105/ml) collected from different concentrations of sanguinarine (0/5/10/30 μmol/l) were placed in the upper chambers with FBS‐free DMEM medium (400 μl), while DMEM medium with 10% FBS (600 μl) was placed in the lower chambers. Non‐invaded cells on the upper surface were wiped by cotton swabs after incubation for 24 hrs, while invaded cells on the lower surface were fixed (30 min., 4% paraformaldehyde) and stained (haematoxylin–eosin, HE). Five fields were selected for counting in each sample. Results were expressed as the average number of migrated cells.
8
Tumor Cell Invasion Inhibition Assay
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A solution of 1×105/ml Tca8113 cells (200 µl free FBS and RPMI 1640 media) in the logarithmic growth phase were plated onto each Transwell chamber (BD Biosciences), following which 10.0 µg/ml R5 or IgG2b was added. The lower chambers were filled with 400 µl RPMI 1640 media containing 0.1% BSA and 16 µg FN, which was used as a chemoattractant. After 24 h in continuous culture 37°C, the Transwell chambers were removed, and the cells in the upper chambers were fixed with 95% ethanol, stained with Giemsa (Beyotime Institute of Biotechnology, Shanghai, China), and counted in 10 microscopic fields (magnification, ×200) per chamber. Each assay was repeated in triplicate, and the invasion inhibitory rate was calculated as follows: Inhibitory rate (%) = (1 - cells in R5-treated group / cells in IgG2b-treated group) × 100%.
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