Cell invasion was tested with real-time invasion assay monitoring using CIM devices and the xCelligence DP system (Roche Diagnostics) [28] (link). Briefly, 4 hrs before the invasion assay, a CIM plate (Roche) was coated with 1∶20 diluted Growth factor reduced Matrigel basement membrane matrix (∼450µg/ml) (BD Biosciences). Then 40,000 cells, untransfected or transfected with empty vector (Ev) or MAEL or PIWIL1 vectors, were seeded into each coated well. Cell activity was followed over a time period of 72 hrs by measuring the impedance signal in the CIM plate. The cell activity was recorded every minute in the first 12 hrs and every 5 mins for the following 12 hrs. Then from 24 hrs onwards until the end of the experiment, cell activity was recorded every 30 mins. In each CIM plate, triplicates of each group were performed to obtain the mean and standard deviation. The experiment was repeated three times.
Cim plate
Manufactured by Roche
Sourced in Germany
The CIM-plates are a type of laboratory equipment designed for cell culture applications. They provide a surface for the cultivation and growth of cells in vitro. The core function of the CIM-plates is to serve as a substrate for cell attachment and proliferation, enabling researchers to study and observe cellular behavior in a controlled laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Rtca system, by Agilent Technologies (3 mentions)
E plate, by Roche (3 mentions)
Anti cxcr2, by R&D Systems (2 mentions)
Xcelligence rtca system, by Roche (2 mentions)
Xcelligence dp system, by Roche (1 mentions)
Matrigel basement membrane matrix growth factor reduced, by BD (1 mentions)
Anti il 8 antibody, by R&D Systems (1 mentions)
Matrigel, by Corning (1 mentions)
Rtca dp, by Roche (1 mentions)
Scrc 1041, by American Type Culture Collection (1 mentions)
Xcelligence rtca dp system, by Roche (1 mentions)
13 protocols using cim plate
1
Real-Time Cell Invasion Assay
2
Two-Chamber Assay for Cancer Cell Migration
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A two-chamber assay (CIM-plate from xCELLigence; Roche 53 (link)) was used to monitor cancer cell migration. The chambers contained serum-free DMEM with or without IL8 (BD Pharminogen) or anti-CXCR2 (R&D Systems) added to both the upper and lower chamber of the assay system. Cancer cells were grown in serum-free DMEM overnight before harvesting for the assay.
3
Two-Chamber Assay for Cancer Cell Migration
4
Real-Time Cell Migration Assay
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The RTCA xCelligence system (Roche Applied Sciences, Almere, the Netherlands), based on cell–electrode substrate impedance detection technology, was used for real-time migration assays. We used CIM plate (Cat# 05665817001, Roche) for migration assay. Briefly, we seeded 4 × 104 cells in serum-free media into each chamber. After all chambers were set up, the CIM plate was put into xCelligence instrument at 37°C, 5% CO2 incubator and cell index was recorded every 15 min intervals. Experiments were performed in triplicate.
5
Assessing Cancer Cell Migration
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Cancer cell migration was assessed by using the OrisTM Cell migration kit (Platypus Technology, Madison, WI), as previously described [38 (link)]. MDA-MB-231- fibroblast or macrophage conditioned media (100 μl) with or without 0.1 uM reparixin (MCE, Monmouth Junction, NJ) and anti-IL-8 antibodies (R&D Systems, 30 μg/ml) was added once the cancer cells had attached. Migration and proliferation assays using CIM-plates (Roche, Indianapolis, IN) and the RTCA system (ACEA Bioscience, San Diego, CA) were performed as previously described [47 (link)].
6
Quantifying HUVEC Migration and Adhesion
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HUVEC migration was assessed, using CIM-plates (Roche) and the RTCA system (ACEA Bioscience); adhesion was assessed using E-plate (Roche) in the RTCA system55 (link). Briefly, the membrane of the top chamber of a CIM-plate was coated with fibronectin by adding 40 µL of 20 µg mL−1 fibronectin dissolved in PBS and incubating at 37°C for 30 min. 180 µL of EGM-2 (complete media for HUVECs) or EBM (serum free media) or MB231-LEC CM was added to the bottom chambers. The equilibrated plate was removed from the incubator and 100 µL of the trypsinized cells (45,000 HUVECs per well) with or without inhibitors were added to the top chamber. After 30 min incubation at room temperature, the stabilized chamber was loaded in the RTCA machine and the cell index was measured continuously for 20 h. Cell indices at 20 h were selected for analysis. ACEA E-plates (Roche Diagnostics) were used to measure the extent of HUVEC adhesion. Briefly, HUVECs (25,000 cells per well) in 100 µL of EGM-2 (complete media for HUVECs) or EBM (serum free media) or MB231-LEC CM were added. After equilibrating at room temperature for 30 min, the E-plate was loaded into the RTCA personal system. Cell indices at 3 h were analyzed.
7
Real-Time Cell Migration and Invasion
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Cell migration and invasion were determined using the xCELLigence RTCA system (Roche). Cells were transfected with the indicated siRNAs for 48 h and starved in a medium without FCS 24 h before the start of the real-time recording. Cells were seeded in 16-well CIM plates (Roche) at the following densities: UM-UC-6 and UM-UC-10 (both at 50,000 cells/well); HT-1376, TCC-SUP-G, JON, and RT-112 (all at 60,000 cells/well); J82 (70,000 cells/well); and CAL-29 (80,000 cells/well). For the cell migration experiments, the membrane between the upper and lower chambers of the CIM plate was left uncoated, whereas for the cell invasion assays, it was coated with 20 µL of Matrigel (Corning (Glendale, AZ, USA)). Matrigel was diluted in a serum-free medium to a concentration of 400 µg/mL and allowed to solidify at 37 °C for 4 h before coating. FCS (10%) was used as a chemoattractant. Cell indices were recorded every 15 min.
8
Quantifying HUVEC Migration and Adhesion
9
Cellular Migration Dynamics Assessed by xCELLigence
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The migration and chemoattractant potential of the cells were measured using an xCELLigence system RTCA DP real-time cell analyzer fitted with CIM plates (05665817001, Roche). The CIM plates have 16-well migration chambers comprising upper and lower chambers separated by a porous (pore size 8 μm) polyethylene terephthalate (PET) membrane in conjunction with microelectrodes. The lower chamber of a 16-well CIM plate was filled with cell culture medium, whilst the upper chamber was seeded with 2.0 × 104 cells in medium. In the xCELLigence assay, three test groups were used; (1) MSCs in the upper chamber alone with PBMCs (1 : 1) in the lower chamber to test directed cell movement without cell-to-cell contact, (2) MSCs in the upper chamber alone and 1% FBS as a negative control in the lower chamber, and (3) PBMCs in the upper chamber alone and 1% FBS as a second negative control in the lower chamber. After equilibration, the analyzer was programmed to scan the membrane every 15 minutes for the first 24 hours and thereafter once an hour.
10
Real-Time Cell Migration Assay
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The migration of the wild-type (WT) and drug conditioned (DC) cell lines was measured using an xCELLigence system RTCA DP real-time cell analyzer (ACEA Biosciences Inc. San Diego, CA) fitted with CIM plates (05665817001, Roche Basel, CH).
These 16 well migration chambers consist of an upper and a lower chamber that are seperated by a porous polyethlene terephtalate (PET) membrane. The 8 um pores allow the cells to migrate from the upper chamber, where 2.0 × 104 cells in FBS-free medium were seeded, to the lower chamber filled with media containing FBS. Every cell migrating to the lower chamber causes a change in the impedance of the xCELLigence system.
The analysis was performed every 15 minutes for the first 24 hours and once every hour thereafter. The experiments were repeated 3 independent times.
These 16 well migration chambers consist of an upper and a lower chamber that are seperated by a porous polyethlene terephtalate (PET) membrane. The 8 um pores allow the cells to migrate from the upper chamber, where 2.0 × 104 cells in FBS-free medium were seeded, to the lower chamber filled with media containing FBS. Every cell migrating to the lower chamber causes a change in the impedance of the xCELLigence system.
The analysis was performed every 15 minutes for the first 24 hours and once every hour thereafter. The experiments were repeated 3 independent times.
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