200µL of basement membrane (Corning Life Science, Shanghai, China) diluted with phosphate buffer saline (PBS) were added to the upper chambers and incubated at 37 °C, 5% CO2 for 2 h to discard unnecessary PBS. The upper chambers for migration did not require any treatment. HeLa and Ca Ski cells were collected and resuspended in serum-free medium and then inoculated into the upper chambers. The lower chambers were filled with culture medium containing 10% FBS. The chambers of HeLa and Ca Ski cells were incubated at 37 °C, 5% CO2 for 8 and 17 h, respectively, then fixed with 4% formalin for 30 min, stained with 0.1% crystal violet for 15 min and finally observed in light microscope (Nikon, CI-L).
Basement membrane
Manufactured by Corning
Sourced in United States
Basement membrane is a thin, sheet-like extracellular matrix that separates epithelial cells from the underlying connective tissue. It provides a structural and functional support for the epithelium and plays a crucial role in tissue organization and cell differentiation.
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Lab products found in correlation
5 protocols using basement membrane
1
Cell Migration Assay Protocol
2
Cell Migration and Invasion Assays
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Cell migration was detected by wound healing assay. HT29 and SW620 cells transfected with si-ACTL8 and si-NC were seeded in six-well plate at a density of 5×105 (link) cells per well, and cultured to 100% confluency before transfection. Then, a wound was created with a pipette tip. After washing the shedding cells, the remaining cells were cultured in medium without FBS. At 0 and 48 hours after injury, images were taken using Nikon Eclipse TS100 Microscope (Nikon, Tokyo, Japan). The assay was independently repeated three times.
To obtain more convincing results, transwell cell invasion assay was performed as follows. After spreading basement membrane (356234; Corning, Corning, NY, USA) in the upper chamber, 1×105 (link) cells/well were incubated with serum-free medium. Medium containing 10% FBS was added into the lower chamber. After 48 hours, cells attached to the lower surface were stained with 0.1% crystal violet (C8470; Solarbio). The migrated cells were counted under a light microscope in five predetermined fields (magnification, ×200). The assay was independently repeated three times.
To obtain more convincing results, transwell cell invasion assay was performed as follows. After spreading basement membrane (356234; Corning, Corning, NY, USA) in the upper chamber, 1×105 (link) cells/well were incubated with serum-free medium. Medium containing 10% FBS was added into the lower chamber. After 48 hours, cells attached to the lower surface were stained with 0.1% crystal violet (C8470; Solarbio). The migrated cells were counted under a light microscope in five predetermined fields (magnification, ×200). The assay was independently repeated three times.
3
Xenograft Model of A549 Lung Cancer
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Four-to-five-week-old BALB/c nude mice were divided into two groups. A549/sh-Ctrl and A549/sh-UPLA1 were prepared as cell suspensions with 3 × 106 cells/100 µL (phosphate-buffered saline : Basement Membrane = 1:1) (Basement Membrane; Corning, NY, USA). The suspensions were administered subcutaneously to nude mice and the tumour growth was observed regularly. After about 40 days, tumour-bearing mice were anesthetised and photographed. Subsequently, tumour peeling was performed. Tumours were weighed and fixed in paraformaldehyde. Ethical approval for the animal experiments was obtained from Shandong Provincial Hospital for Animal Experimentation (No. 2019122).
4
Transwell Migration Assay Protocol
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A transwell migration assay was performed using an 8μm pore polycarbonate membrane 24-well transwell unit (Corning Incorporated, Costar, USA, ref 3422). The transwell unit was treated with Basement Membrane (Corning Incorporated, Matrigel Matrix, USA, ref 356234) preliminarily according to instruction. filling the lower compartment with 10% FBS medium as a chemoattractant. In the meantime, after overnight starvation, resuspended cells were plated in the upper compartment with serum-free medium. After incubation for 24 hours at 37°C with 5% CO2, the cells in the upper compartment were completely wiped off by gentle swabbing. We used crystal violet to stain the cells that had migrated to the lower surface of the membranes for 10 minutes. Then, we took photos and counted invading cells (using Image J to calculated) in five 200× magnifying fields.
5
Cell Migration Assay Protocol
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The density of cell suspension was adjusted to 5×104/ml. Next, 2 ml cell suspension was added into the transwell, a 6-well plate was cultured for 12 hr in an incubator, and the basement membrane (Corning Company, USA) was taken out. After that, cotton swabs were used to wipe out the non-migrated cells from the transwell. Meanwhile, migrated cells were fixed for 15-20 min with 95% alcohol, stained for 10 min by hematoxylin, and observed under an optical microscope.
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