Migration and invasion assays were performed in 24-well cell culture chambers (Costar 3422; Corning Inc., Corning, NY, USA), and the lower and upper chambers were separated by a polycarbonate membrane (8-μm pore size). About 4 × 104 cells stimulated without or with NEC-1 and TNFα were seeded on the upper chamber with DMEM without FBS. Moreover, the upper chambers were precoated with Matrigel (BD, Franklin Lakes, USA) for the invasion assay rather than for the migration assay. DMEM containing 10% FBS was added to the lower chamber. After incubation for 24–48 h at 37°C with 5% CO2, we removed the cells remaining on the upper membrane with cotton wool. At the same time, the cells on the other side of the membrane were fixed with methanol and stained with 0.1% crystal violet solution. Finally, a microscope (Olympus, Tokyo, Japan) was used to observe the cells.
Costar 3422
Manufactured by Corning
Sourced in United States
The Costar 3422 is a 96-well flat-bottom cell culture plate made of polystyrene material. It is designed for various cell culture applications in life science research, including cell proliferation assays, drug screening, and cytotoxicity studies.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (6 mentions)
Matrigel solution, by BD (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
High glucose dmem, by Thermo Fisher Scientific (2 mentions)
Evos microscope, by Thermo Fisher Scientific (2 mentions)
Difco 0.5x nb, by BD (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Crystal violet, by Solarbio (1 mentions)
Paraformaldehyde (pfa), by Solarbio (1 mentions)
Irigenin, by Selleck Chemicals (1 mentions)
F2518, by Merck Group (1 mentions)
Ecm001, by Merck Group (1 mentions)
Ab182981, by Abcam (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Ae30 31, by Motic (1 mentions)
Giemsa, by Merck Group (1 mentions)
Pannoramic midi, by 3DHISTECH (1 mentions)
Three step stain set diff quik, by Sysmex (1 mentions)
Nis elements software, by Nikon (1 mentions)
Formaldehyde, by Beyotime (1 mentions)
53 protocols using costar 3422
1
Cell Migration and Invasion Assay
2
Cell Migration Assay Protocol
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Migration assays were performed using 24-well plates with transwell permeable supports of 6.5 mm insert and a polycarbonate membrane with an 8 µm pore size (Costar 3422, Corning Inc., Corning, NY, USA). Cells were seeded in the upper chamber at 1 × 104 cells/mL in 0.1 mL of serum-free RPMI-1640 media. A volume of 0.8 mL of media supplemented with 10% FBS was placed in the bottom well as a chemo-attractant. After incubation for 24 h at 37 °C in an atmosphere containing 5% CO2, migrated cells on the lower surface were stained using crystal violet and counted under a light microscope.
3
Transwell Migration Assay for miR-885-5p
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Migration assays were performed in triplicate using a 24-well Transwell chamber (Costar 3422; Corning Inc., Corning, NY, USA). MG-63 cells transfected with miR-885-5p-up, miR-885-5p-down and NC, respectively, were seeded in the upper chamber at 2×104 cells per well in serum-free DMEM. DMEM supplemented with 10% FBS was added to the bottom well at a volume of 0.6 ml. Following incubation for 24 h, the cells on the top filter were gently removed with cotton swabs and cells that had migrated to the lower surface were fixed with methanol for 30 min at room temperature and stained with 0.5% crystal violet for 30 min at room temperature. The values for migration were obtained by counting three fields under a light microscope. The experiment was repeated three times over multiple days.
4
Evaluating Antibacterial Hydrogel Efficacy
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To examine whether the hydrogel system loaded with the peptidomimetic is able to counteract bacterial migration across surfaces, the upper chambers of a trans-well plate (Costar 3422®, Corning Corporation, Corning, NY, USA) were coated with control saline solution, HA-BDDE hydrogel or HA-BDDE hydrogel loaded with (ri)-r(P)ApoBSPro, as described by Xiaojuan Li et al. [33 (link)]. Following coating, methicillin-resistant S. aureus (MRSA WKZ-2) and E. coli ATCC 25922 bacterial cells were diluted to 4 × 106 CFU/mL in Difco 0.5X NB (Becton-Dickinson, Franklin Lakes, NJ, USA), and plated into the previously coated upper chambers. The medium in the lower wells was then analyzed at defined time intervals, in order to monitor bacterial cells’ migration from the upper to the lower wells. The number of migrated bacteria was quantified by diluting and plating each sample on TSA. Following an incubation of 24 h at 37 °C, the number of colonies was counted. The experiment was performed in three independent replicates.
5
Transwell Assay for Cell Migration
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Transwell migration assays were performed using a 24-well chamber (Costar 3422; Corning Inc., Corning, NY, USA). A549 cells (2 × 104) were suspended in 100 μl of serum-free medium in the upper chamber. Medium containing 10% FBS was added to the lower chamber. The cells could migrate for 24 h to allow cell migration through the membrane. The membrane was fixed in 4% paraformaldehyde for 30 min and stained with crystal violet. Cells on the lower side of the membrane were counted37 (link).
6
Transwell Assay for Cell Invasion and Migration
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MHCC-97H cell were treated with indicated concentrations of anti-tumor agents and analyzed by transwell assays performed in 24-well plates chamber (Cat. No.: Costar 3422, Corning, Lowell, MA, USA) fitted with a polyethylene terephthalate filter membrane with 8-μm pores. The invasion-transwell or migration-transwell was performed following the methods described by Ji et al. and Liang et al.34 (link), 35 (link).
7
Transwell Invasion and Scratch Wound Assays
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We used a 24‐well chamber to perform transwell invasion assays (Costar 3422; Corning Inc., Corning, NY). SMCs were cultured in the upper chamber into DMEM. In the lower chamber, additional 10% FBS was added to DMEM. After 24 hours, SMCs were allowed to migrate from the upper chamber to the underside of the membrane. Migrated SMCs on the lower membrane were counted using an Olympus light microscope and analyzed using the Image J. For scratch wound assay, we used a 100‐μL pipette tip to scratch SMCs and cultured for 36 hours. We performed scratch wound assay in the presence of 25 μg/mL Mitomycin C in order to inhibit SMC proliferation. SMCs were visualized using a microscope and captured images were assessed using Image‑Pro Plus software.
8
Transwell Assay for Cell Migration
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Transwell migration assays were performed using a 24-well chamber (Costar 3422; Corning Inc., Corning, NY, USA). The lower and upper chambers were partitioned by a polycarbonate membrane (8-μm pore size). Lung cancer cells (5 × 103) were seeded into RPMI-1640 without FBS in the upper chamber. RPMI-1640 containing 10% FBS was added to the lower chamber. The cells were allowed to migrate for 36 h at 37 ˚C in a humidified atmosphere containing 5% CO2. Cells remaining on the upper side of the membrane were removed using PBS-soaked cotton swabs. The membrane was then fixed in 4% paraformaldehyde for 20 min at 37 ˚C and then stained with crystal violet. Cells on the lower side of the membrane were counted under an Olympus light microscope (Olympus, Tokyo, Japan).
9
Transwell Migration Assay with Doxorubicin
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Migration or invasion assays were performed using 24-well plates inserted by 8-μm pore size transwell filter insert (Costar 3422 [Corning, Corning, New York, USA]) with or without pre-coated diluted Matrigel (1:8) (Becton Dickinson and Co., Franklin Lakes, NJ, USA). 5 × 104 IOMM-Lee cells with Serum-free medium were placed into the upper chamber, and 500 μL medium containing 30% FBS was added into the bottom chamber subsequently. Doxorubicin was added at the concentration of IC50. DMSO (Sigma-Aldrich, St. Louis, MO, USA) was used as the negative control group. After incubation in 37 °C for 24 h, cells on the underside of membrane were immobilized about 40 min with 4% Paraformaldehyde (Solarbio, Beijing, China) and stained about 20 min with 0.4% crystal violet (Solarbio, Beijing, China). Then penetrated cells were counted in five random fields under the microscope. Migrated areas of cells were calculated by ImageJ (version2.3.0, National Institutes of Health, Bethesda, MD, USA).
10
Transwell Assays for Cell Migration and Invasion
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Migration assays were performed using a 24-well Transwell chamber system (Costar 3422; Corning Incorporated, Corning, NY, USA). Cells were seeded in the upper chambers at density of 1×104 cells/ml in 0.1 ml serum-free DMEM. The lower chambers were filled with 0.8 ml DMEM with 10% fetal bovine serum. Following incubation for 24 h at 37°C in a 5% CO2 atmosphere, the cells that migrated through the membrane were fixed with methanol for 15 min, stained with crystal violet stain for 10 min at room temperature and counted using a microscope at ×200 magnification.
Invasion assays were performed using a 24-well Transwell chamber coated with Matrigel (30 µl per filter; BD Biosciences), according to the manufacturer's protocol. Cells were seeded on the top of Matrigel-coated invasion chambers in serum-free DMEM (2×104 cells/well) and DMEM containing 10% fetal bovine serum was added to the lower chambers. Subsequent to incubation for 24 h at 37°C in a 5% CO2 atmosphere, the cells that invaded through the membrane were fixed with methanol for 15 min, stained with crystal violet stain for 10 min at room temperature and counted using a microscope at ×200 magnification.
Invasion assays were performed using a 24-well Transwell chamber coated with Matrigel (30 µl per filter; BD Biosciences), according to the manufacturer's protocol. Cells were seeded on the top of Matrigel-coated invasion chambers in serum-free DMEM (2×104 cells/well) and DMEM containing 10% fetal bovine serum was added to the lower chambers. Subsequent to incubation for 24 h at 37°C in a 5% CO2 atmosphere, the cells that invaded through the membrane were fixed with methanol for 15 min, stained with crystal violet stain for 10 min at room temperature and counted using a microscope at ×200 magnification.
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