Monocytes were isolated from the femur and tibias of C57Bl/6 mice by use of a bone marrow monocyte isolation kit (MACS Miltenyi Biotec, 130–100629). Migration experiments were carried out with CIM-16 plates and an xCELLigence RTCA-DP instrument (ACEA, San Diego, USA) as previously described [76 ]. Chemoattractants were made to desired concentrations and loaded into the lower wells of the CIM-16 plate. Upper wells were filled with chemotaxis buffer and plates equilibrated for 30 min at RT. Elicited macrophages were resuspended in chemotaxis buffer incubated at 37°C, 5% CO2 for 1 h prior to assay commencement. Cell suspensions were placed into the wells of the upper chamber, and the assay performed over 8 h (5 s data points). Adhesion assays were performed with E-plates and an xCELLigence RTCA-DP instrument (ACEA, San Diego, USA). Monocytes were isolated from platelet deficient or competent mice, and adhesion monitored over 8 h (2 min data points).
Xcelligence rtca dp instrument
Manufactured by Agilent Technologies
Sourced in United States
The XCELLigence RTCA DP instrument is a real-time cell analysis (RTCA) system designed to monitor cell proliferation, cytotoxicity, and cell adhesion in real-time. The instrument uses an electronic sensor technology to measure changes in electrical impedance, which is proportional to the number, attachment, and morphological changes of cells on specialized microplates.
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Lab products found in correlation
Cim plate 16, by Agilent Technologies (4 mentions)
Crystal violet, by Solarbio (3 mentions)
E plate 16, by Agilent Technologies (3 mentions)
Cim plate, by Agilent Technologies (2 mentions)
Matrigel, by Corning (2 mentions)
Cytotox 96 kit, by Promega (1 mentions)
Mtt reagent, by Merck Group (1 mentions)
Cck 8 reagent, by BestBio (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
Celltiter 96 aqueous non radioactive cell proliferation assay kit, by Promega (1 mentions)
I5029, by Merck Group (1 mentions)
Winnonlin software, by Pharsight (1 mentions)
E16 plate, by Agilent Technologies (1 mentions)
Polycarbonate membrane, by BD (1 mentions)
Rtca software 2, by Agilent Technologies (1 mentions)
E plate, by Agilent Technologies (1 mentions)
Accutase, by Merck Group (1 mentions)
E plate view 16, by Agilent Technologies (1 mentions)
Xtt cell proliferation assay kit, by American Type Culture Collection (1 mentions)
Rtca software 1, by Agilent Technologies (1 mentions)
60 protocols using xcelligence rtca dp instrument
1
Isolation and Migration of Murine Monocytes
2
Real-Time Cell Proliferation Assay
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xCELLigence RTCA DP instrument (ACEA Biosciences Inc., San Diego, CA, USA) was placed in the humidified incubator at 37 °C and 5 % CO2 atmosphere. Cell proliferation experiments were carried out using E-plates according to manufacturer protocol. Cells were seeded into E-16 plate at a density 2×104 in 100ul per well and experiment was running for 96 h. Each experiment was performed in triplicate. Data was analyzed using RTCA software and Slope was calculated every 12 h.
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xCELLigence RTCA DP instrument (ACEA Biosciences Inc., San Diego, CA, USA) was placed in the humidified incubator at 37 °C and 5 % CO2 atmosphere. Cell proliferation experiments were carried out using E-plates according to manufacturer protocol. Cells were seeded into E-16 plate at a density 2×104 in 100ul per well and experiment was running for 96 h. Each experiment was performed in triplicate. Data was analyzed using RTCA software and Slope was calculated every 12 h.
3
Real-Time Monitoring of Cell Proliferation and Adhesion
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A 1,000-µL cell suspension containing 2,000 cells, which were in the logarithmic phase, was obtained from the cells cultured in the 24-well plates. The GC-030-35 cells were placed in the xCELLigence® RTCA DP instrument (ACEA Biosciences Inc, San Diego, CA, USA) which used electrical impedance to monitor cell morphology, quantify cell proliferation, and cell attachment, every hour for 8 days. From the growth curve, the cell population doubling time (PDT), the cell index, and integration times were calculated according to the formula: PDT = (T − T0) lg2/(lgNt − lgN0), where PDT represents the cell PDT, and T0 and T represent the starting time and end of the cell culture.
The cell index was used to indicate cell impedance, which reflected the number of adherent cells. To explore the plating efficiency of cells, 100, 500, 1,000, and 2,000 single-cell suspensions were placed in 60-mm diameter culture dishes and cultured for 9 days. The ratio of clone number to total inoculated cell number was calculated, and ≥50 cells were considered to represent plating efficiency.
The cell index was used to indicate cell impedance, which reflected the number of adherent cells. To explore the plating efficiency of cells, 100, 500, 1,000, and 2,000 single-cell suspensions were placed in 60-mm diameter culture dishes and cultured for 9 days. The ratio of clone number to total inoculated cell number was calculated, and ≥50 cells were considered to represent plating efficiency.
4
Real-Time Cell Growth Monitoring
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Cell growth and proliferation were monitored in real time using an xCELLigence RTCA DP instrument (ACEA Biosciences, San Diego, USA) for 72 h. For the colony formation assay, a total of 1000 SW1116 and HT29 cells were seeded into each well of a six-well plate and were maintained in DMEM containing 10% FBS for ~ 2 weeks. The medium was replaced every 3 days. Then, the colonies were fixed with methanol and were stained with 0.1% crystal violet (Solarbio, Beijing, China). The numbers of stained colonies were determined by counting.
5
Real-time Analysis of GBM Cell Migration
6
Real-time Analysis of GBM Cell Migration
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Transwell-migration of the retrospective patient-derived primary cells were monitored using the xCELLigence RTCA DP instrument (Acea Biosciences, Inc.) according to the manufacturer’s protocol using a CIM-plate 16 chambers32 (link). These plates have chambers that are similar to Boyden chambers; they consist of an upper chamber where the GBM cells are seeded in serum-free DMEM/F12, a microporous polyethylene terephthalate (PET) membrane with an average pore diameter of 8 μm (through which GBM cells migrate to the lower chamber), electrodes directly below this membrane, and a lower chamber which is filled with DMEM/F12 containing Gem21 Neuroplex supplemented with EGF and FGF as chemoattractant. 4 × 104 cells per well were seeded into the upper chamber of a CIM-plate 16 chambers. The extent of GBM cell migration was monitored in real time as cell index for 48 h at 15 min intervals in a humidified incubator maintained at 37 °C and 5% CO2 by measuring changes in electrical impedance with electrodes that are attached directly underneath the PET membrane. Each patient-derived primary GBM specimen was run in triplicate for each experiment.
7
Real-Time Cytotoxicity Monitoring in Culture
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The xCELLigence RTCA DP instrument (ACEA Bioscience) monitors cell impedance in real time. Cells were inoculated in E‐plate 16 at a density of 10 000 cells per well and incubated overnight before the addition of cytotoxic agents. The cell index was monitored for one week.
8
Cell Migration Assay using xCELLigence
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Migration assays were performed using an xCELLigence RTCA DP instrument (ACEA Biosciences, Inc.), and cell invasion and migration plates (CIM-Plate®; ACEA Biosciences, Inc.). Briefly, 5×104 cells/well in 150 µl serum-free media were seeded on the upper electronically integrated Boyden chamber above a membrane with a pore size of 8-µm. The lower chamber contained 160 µl media supplemented with 10% FBS as a chemoattractant. The CI was measured every 15 min for 18 h (as an indication of the rate of cell migration onto the underside of the top chamber). All experiments were performed in triplicate, with at least three independent repeats.
9
Cytotoxicity and Cell Viability Assays
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To measure the amount of dead or growing cells, we used CytoTox96 kit (Promega, UK) and 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (Sigma Aldrich, USA). C6 or B16 cells were placed in 96-well plates and after overnight cultivation AEAC was added in the concentration indicated. Twenty-four hours later, cytotoxicity and the viability value were measured. Acridine orange staining was used to measure the number of apoptotic cells. The xCelligence RTCA DP instrument (ACEA Biosciences®, Inc, USA) was used to measure the growth dynamics of C6 or B16 cells that were exposed to doxorubicin and AEAC.
10
Quantifying Cell Migration and Invasion
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A549 cells were seeded in tetraplicate at a density of 2 × 104 cells/well in the upper chamber of CIM-Plate either uncovered (migration assay) or covered with Matrigel (10 µL per well, diluted 1:40 with cold serum-free DMEM) (invasion assay) in serum-free DMEM in the presence or absence of SM (0.5 µM) and TGF-β (50 ng/mL). In order to stimulate motility of the cells, DMEM supplemented with 10% FBS as chemoattractant was placed to the lower chamber of CIM-Plate. Thereafter, the electrical impedance (cell index) of sensor electrodes mounted at the lower side of porous membrane separating the upper and lower chambers of the plate was further measured using xCELLigence RTCA DP instrument (ACEA Biosciences, San Diego, CA, USA) every 1 h for 48 h.
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