A 96-well plate with 3000 bone marrow-derived mesenchymal stem cells (BMSCs) per well were seeded in different concentrations of deferoxamine (DFO), and cell proliferation was determined by measuring the area of the cells using the Incucyte HD imaging system (Essen BioScience, Ann Arbor, MI, USA). DFO was purchased from Novartis Pharma (Tokyo, Japan).
Incucyte hd imaging system
Manufactured by Sartorius
Sourced in United States, Japan
The Incucyte HD imaging system is a live-cell analysis platform that enables real-time, automated monitoring and quantification of cell growth, morphology, and behavior within a controlled environment. The system combines advanced optics, environmental controls, and powerful image analysis software to provide users with detailed insights into their cell cultures.
Automatically generated - may contain errors
Lab products found in correlation
Hela cells jcrb9004, by Japanese Collection of Research Bioresources Cell Bank (1 mentions)
Calcusyn software program, by Biosoft (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Cck 8 solution, by Dojindo Laboratories (1 mentions)
H2dcfda, by Thermo Fisher Scientific (1 mentions)
6 protocols using incucyte hd imaging system
1
Deferoxamine Impacts BMSC Proliferation
2
MSC Proliferation Assay with DFO
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To measure proliferation, 1,000 MSCs were seeded in each well of a 96-well plate, DFO was then added at different concentrations, and the cellular area was evaluated with an IncuCyte HD imaging system (Essen BioScience, London, UK). DFO was purchased from Novartis Pharma K.K. (Tokyo, Japan).
3
MSC Proliferation Measurement with AAP
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MSCs were seeded into each well of a 96-well plate (3000 cells/well), and AAP (Tokyo Chemical Industries, Ltd. Tokyo, Japan) was added at indicated concentrations. Proliferation was measured using the IncuCyte HD Imaging System (Essen BioScience, Tokyo, Japan).
4
Establishing DFO-resistant Cell Lines
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HeLa cells (JCRB9004) and Huh7 cells (JCRB0403) were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Japan) supplemented with 10% fetal bovine serum (SAFC, MO, USA). The DFO-resistant cell lines were established by gradually increasing the DFO concentration (started from 3 μM) over a duration of approximately 6 months. Cells (2000~6000/well) were seeded in 96-well plates and treated with different concentrations (0~100 μM) of DFO, and cell proliferation was determined by measuring the area of the cells using the Incucyte HD imaging system (Essen BioScience, Ann Arbor, MI). The combination activity of the drugs was estimated with the CalcuSyn software program (Biosoft, Ferguson, MO). Briefly, this program determines the combination index (CI), a quantitative measure of the degree of drug interactions. The information related to drugs is provided in Supplemental Table 1 .
5
Evaluating Serum Effects on MSC Proliferation
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MSCs were plated at 2,000 cells/well in 96-well plates and then treated with different serum concentrations from ACLF patients and HC volunteers. Ten microliters of cell counting kit 8 (CCK-8) solution (Dojindo, Kumamoto, Japan) was added at the indicated time points on days 1, 3, 5, and 7. Proliferation was measured using the IncuCyte HD imaging system (Essen BioScience, Tokyo, Japan) by measuring the optical density value at 450 nm. The results are expressed as the percentage of proliferation±standard error of the mean and normalized to 100% as the initial number of cells plated.
6
Measuring ROS Using H2DCFDA and Incucyte
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Reactive oxygen species were measured using 2',7'dichlorodihydrofluorescein diacetate (H 2 DCFDA, Life Technologies) and fluorescence intensity was measured using the Incucyte HD imaging system (Essen Bioscience, Ann Arbor, MI, USA). The Incucyte HD imaging system was used to measure the intensity of H 2 DCFDA fluorescence in each captured image at different times. We evaluated the changes in reactive oxygen species (ROS) by ephedrine at different times. After treatment with ephedrine and 5 μM H 2 DCFDA, fluorescence intensity was measured every 30 min.
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