For cell preparation, 3×104 cells were seeded into a two-well silicone culture insert (ib80209, Ibidi) in a 35 mm dish and cultured overnight. To stop cell proliferation, cells were treated with 10 µg/ml mitomycin C-containing medium for 2 h, and then the culture inserts were removed. Cells were washed twice with PBS and cultured in a medium with or without 10 ng/ml TGF-β for 24 h. Cell images were taken at 0 h, 12 h and 24 h time points.
Ib80209
Manufactured by Ibidi
Sourced in Germany, France
The Ib80209 is a lab equipment product offered by Ibidi. It serves as a core function for laboratory applications, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
3 protocols using ib80209
1
Cell Migration Assay with TGF-β
2
Cell Migration Assay with CdM
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MS1 cells were seeded at 2.1 × 104 cells per well into silicon culture inserts with two individual wells (#ib80209, Ibidi, Munchen, Germany) on cell culture slides (Ibidi) for 24 h. The culture inserts were removed, and cells were incubated with DMSO- or GW4869-treated CdM. Images were acquired using a BZ-X710 microscope (Keyence, Osaka, Japan) at 0, 24 and 36 h after cell migration, and were analyzed with Fiji software.
3
Calu-3 Cell Migration and Proliferation
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To assess migration and proliferation properties of shRNA-or CRISPR-modified Calu-3 cells, untransduced, shAlter, sh1+sh2, CTL and TSJB1 cells (5×10 4 ) were seeded in silicone culture-inserts (IB-80209, Ibidi, Biovalley, Marne-la-Vallée, France) and cultured until confluence at which point the chambers were removed (t=0h). Gap closure was monitored 15 hours later (t=15h). Pictures were taken at t=0h and t=15h using the EVOS FL microscope (AMG, Bothell, WA) and the entire gap surface was reconstructed using Photoshop software. The gap area was measured as a pixel number using ImageJ software.
To quantify the number of proliferating cells, inserts were fixed in 4% paraformaldehyde for 15min and then processed in the following solutions: PBS-Triton 0.1% for 10min, NH4Cl 0.5M for 15min and PBS-BSA 2% for 15min. Calu-3 cells were then incubated with the monoclonal anti-Ki-67 antibody (1:100 dilution, mib1, DAKO, Les Ulis, France) followed by a goat anti-mouse Alexa 568 antibody (1:1500, Jackson ImmunoResearch Laboratories). DAPI was used to stain nuclei. Ki-67 and DAPI-positive cells were counted using ImageJ software.
To quantify the number of proliferating cells, inserts were fixed in 4% paraformaldehyde for 15min and then processed in the following solutions: PBS-Triton 0.1% for 10min, NH4Cl 0.5M for 15min and PBS-BSA 2% for 15min. Calu-3 cells were then incubated with the monoclonal anti-Ki-67 antibody (1:100 dilution, mib1, DAKO, Les Ulis, France) followed by a goat anti-mouse Alexa 568 antibody (1:1500, Jackson ImmunoResearch Laboratories). DAPI was used to stain nuclei. Ki-67 and DAPI-positive cells were counted using ImageJ software.
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