Transwell inserts for 24-well plates (8 µm; Corning, Inc.) were coated with pre-diluted Matrigel (1:8; BD Biosciences) at 37°C for 30 min. HCT116 cells, at a density of 5×105 cells in 200 µl DMEM, were seeded into the Matrigel-coated upper chamber, while the lower chamber was filled with 400 µl DMEM supplemented with 10% FBS. Following incubation at 37°C for 24 h, the cells on the lower surface of the membrane were fixed with 10% formaldehyde and stained with 0.1% crystal violet solution both for 15 min at room temperature. The number of invading cells was counted under a light microscope (Nikon Corporation; magnification, ×100) and cells were analyzed using ImageJ (v. 1.8.0; National Institutes of Health).
Transwell inserts for 24 well plates
Manufactured by Corning
Sourced in United States
Transwell inserts are permeable supports designed for use with 24-well culture plates. They provide a semi-permeable membrane that allows the exchange of media, nutrients, and other substances between the upper and lower chambers of the well.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (2 mentions)
Anti β actin antibody, by Abcam (1 mentions)
Monoclonal anti e cadherin, by Abcam (1 mentions)
Anti vimentin monoclonal antibody, by Cell Signaling Technology (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Mitomycin c, by Merck Group (1 mentions)
Matrigel coated membrane, by BD (1 mentions)
5 protocols using transwell inserts for 24 well plates
1
Transwell Invasion Assay for HCT116 Cells
2
Monoclonal Antibody-based Protein Detection
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Monoclonal anti-HIF-1α antibody (RRID:AB_2835328) was purchased from Affinity (USA), monoclonal anti-E-cadherin (RRID:AB_731493) and anti-β-actin antibodies (RRID:AB_306371) were obtained from Abcam (UK), and monoclonal anti-vimentin antibody (RRID:AB_10695459) was purchased from Cell Signaling Technology (CST, USA). Secondary anti-rabbit immunoglobin G (IgG) antibodies were obtained from Santa Cruz (USA). Neofect™ DNA transfection reagent was purchased from Neofect Biotechnologies (China). T-25-cm2 flasks for cell culture were purchased from Bever (USA) and Transwell inserts for 24-well plates (6.5 mm diameters and 8.0 µm diameter filters) were purchased from Corning (USA). Sealed hypoxia incubator chambers were obtained from Thermo Fisher Scientific (USA). Other chemicals of analytical grade were obtained from Sigma-Aldrich (USA).
3
Modulation of SCAP-PBMC Interactions
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SCAP 5 × 104 were seeded in wells of a 24-well plate or into a transwell inserts for 24-well plates (0.4 μm) (Corning, NY, USA) in RPMI medium +10% HS (ThermoFischer Scientific). SCAP were stimulated with LPS (1 μg/mL) for 24 h, before application of LXA4 (1 to 100 nM), and/or WRW4 (10 μM), while control SCAPs were left untreated. SCAP were washed twice in PBS with 10% Human Serum, treated with mitomycin C (Sigma-Aldrich) at the final concentration of 25 μg/mL for 30 min, and then washed 4 times with PBS + 10% Human Serum. Human peripheral blood mononuclear cells (PBMC) (AllCells, Alameda, CA, USA) in dilution ratios 1:1, 1:5 and 1:10 were stimulated with 5 μg/mL phytohemagglutinin (PHA) (Sigma-Aldrich) and immediately added to the wells containing SCAP. Negative controls (PBMC only) and positive controls (PBMC + PHA) were used. Results of MLR were evaluated after five days of cultivation.
4
Invasion Assay for ccRCC Cells
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The invasion of ccRCC cells was evaluated by transwell inserts for 24‑well plates containing 8‑µm pores (Corning Inc., Corning, NY, USA) with a Matrigel-coated membrane (BD Biosciences, San Jose, CA, USA). Cells (1 × 105) in serum‑free DMEM were seeded into the upper chamber of the transwell insert. In the bottom chamber, a total of 800 µl DMEM supplemented with 20% FBS was served as a chemoattractant. After 48 h of incubation in a 37˚C humidified incubator with 5% CO2, non-invasive cells on the upper surface were carefully removed with a cotton swab, whereas the invasive cells were fixed with 4% paraformaldehyde for 30 minutes and stained with 0.1% crystal violet for 30 minutes at room temperature. The numbers of invasive cells were counted under a microscope (× 200 magnification) in five random fields. The experiment was performed three times.
5
Matrigel-coated Transwell Invasion Assay
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Transwell inserts for 24-well plates (8 µm; Corning, Inc.) were coated with prediluted Matrigel (1:8; BD Biosciences) at 37˚C for 30 min. The cells were washed twice in PBS and then suspended in serum-free DMEM. A total of 5x105 cells in 200 µl medium were placed into the Matrigel-coated upper chamber and the lower chamber was filled with 400 µl DMEM with 10% FBS. Following incubation at 37˚C for 24 h, the non-invading cells in the upper chamber were gently removed. The cells on the submembrane surface were stained with 0.5% crystal violet for 15 min at room temperature and observed under a light microscope (magnification, x100; Nikon Corporation) and images were captured.
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