MUSE cell analysis assay kit (Millipore, USA) that contains PI and RNase A premixed reagent was used for cell cycle analysis. MDA-MB-231 and MCF-7 cells were seeded at a density of 5 × 105 cells/mL in complete DMEM high glucose (Nacalai, Japan) in T25 flasks (SPL Life Sciences, Korea) and incubated for 24 h at 37°C in a humidified incubator (ESCO, Singapore) with 5% CO2 and 95% air. Cells were treated with different doses of 3F1: e.g., IC50, lower than IC50, higher than the IC50 to evaluate whether the compound 3F1 consistently affected the cell cycle of these breast cancer cells. Treated and control cells were incubated for 24 h at 37°C in a humidified incubator supplied with 5% CO2 and 95% air followed by fixation and staining of the cells by manufacturer instructions of the kit. Analysis was done using the MUSE cell analyzer (Merck-Millipore, USA) followed by ModFit LT version 5 to generate the histograms and the percentage of cells in each cell cycle phase.
Humidified incubator
Manufactured by Esco Lifesciences
Sourced in Singapore
The Humidified Incubator is a laboratory equipment designed to maintain a controlled environment for cell culture and other biological applications. It provides a temperature and humidity-regulated chamber to support the optimal growth and development of cells and organisms.
Automatically generated - may contain errors
2 protocols using humidified incubator
1
Cell Cycle Analysis of Breast Cancer Cells
2
In Vitro Cell Culture and Isolation
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HeLa, A375, HepG2, A549 (all of cancer cell lines) and WRL-68 (of normal liver) cells were collected from National Centre for Cell Science, India. The cells were maintained in the humidified incubator (ESCO, Singapore) with ambient oxygen and 5% carbon dioxide level at 37°C. Cells were cultured in DMEM with 10% heat-inactivated FBS and 1% PSN. Cells were harvested with 0.025% Trypsin-EDTA in phosphate buffer saline (PBS), were plated at required cell numbers and allowed to adhere for required time before treatment.
Peripheral blood mononuclear cells (PBMC; normal blood cells from healthy mice) were immediately isolated by gradient centrifugation in Ficoll-Hypaque by a standard method and washed twice with phosphate buffered saline (PBS) and once more with RPMI-1640.
Peripheral blood mononuclear cells (PBMC; normal blood cells from healthy mice) were immediately isolated by gradient centrifugation in Ficoll-Hypaque by a standard method and washed twice with phosphate buffered saline (PBS) and once more with RPMI-1640.
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