The H9c2 cell line, derived from rat atrial cardiomyoblasts, was purchased from the American Type Culture Collection (ATCC; Manassas, VA) and maintained in high glucose DMEM (Dulbecco’s Modification of Eagle’s Medium), supplemented with 10% bovine calf serum and 1% penicillin–streptomycin solution at 37° C with 5% CO2. Cultured adherent H9c2 cells were trypsinized and pelleted by centrifugation at 500×g for 5 minutes at 4°C and cells were washed twice by suspending in complete DMEM; cells were counted using a Z1 COULTER COUNTER® Cell and Particle Counter (Beckman Coulter Inc. Brea CA 92821). For cell viability assays, cell pellets were resuspended at 1×105 cells/ml in DMEM and 100 μL/well were seeded in 96-well plates and allowed to recover for 6-8 h before pretreatment with 2.5 μM SFN or vehicle for 12-14 h. Vehicle- or SFN-pretreated cells were subsequently treated with 5 μg/ml DOX or vehicle for an additional 16-18 h and analyzed for viability by the MTT assay using the CCK-8 kit (Dojindo, Rockville, Maryland) [43 ].
Z1 coulter counter cell and particle counter
Manufactured by Beckman Coulter
Sourced in United States
The Z1 COULTER COUNTER® Cell and Particle Counter is a laboratory instrument designed to count and measure the size of particles, cells, and other suspended matter in a sample. It operates based on the Coulter principle, which detects and measures changes in electrical impedance as particles pass through a small aperture.
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4 protocols using z1 coulter counter cell and particle counter
1
Cardiomyoblast Cell Viability Assay
2
In Vitro Cell Proliferation and Apoptosis Assay
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In vitro cell proliferation rate was determined by monitoring the cell number for 7–9 days in cell culture. The cells in each condition were seeded at 3 × 104 cells/well in a 12-well plate or 1 × 104 cells/well in 24-well in triplicate and incubated in a 37 °C humidified CO2 incubator with the drug or vehicle containing medium refreshed every other day. The number of cells was determined by Z1 COULTER COUNTER® Cell and Particle Counter (Beckman Coulter). For the apoptosis assay, cells were seeded at 3 × 105 cells/well into a six-well plate in triplicate. After 24 h, cells were subjected to starvation using 2% fetal bovine serum medium for 48 h and then followed by flow cytometry analysis. The cell apoptosis assay was determined according to the manual of FITC Annexin V Apoptosis Detection Kit I (BD Biosciences). Data were analyzed by FlowJo software (FlowJo).
3
Evaluating miR-148a and HOTAIR in GC Cells
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Approximately 5 × 104 GC cells were plated in 24-well plates and then transfected with miR-148a mimics or inhibitors or si-HOTAIR or negative control. After 24, 48. and 72 h, the cell numbers were detected by using a Z1 COULTER COUNTER Cell and Particle Counter (Beckman Coulter, Fullerton, CA, USA). For cell cycle analyses, the GC cells were seeded in 6-well plates and then transfected with si-HOTAIR. 48 h later, the transfection, cells were fixed in 70% ethanol at 4°C for 24 h and stained with propidium iodide (Beytime, Beijing, China). The cell cycle distribution was assessed by flow cytometry (BD FACS Calibur, American).
4
Assessing MDA-MB-231 and MCF7 Cell Viability and Colony Formation
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Cultured adherent MDA-MB-231 or MCF7 cells were trypsinized and pelleted by centrifugation at 500 g for 5 min at 4 °C and washed twice by suspension in complete DMEM; cells were counted using a Z1 COULTER COUNTER cell and particle counter (Beckman Coulter Inc., Brea, CA, USA). For cell viability assays, cells were seeded at 1 × 105 cells/mL in DMEM and 100 µL/well in 96 well plates and allowed to recover for 16–18 h. The next day, one set of cells was transfected with 0.1 µg/well CAS, R508, or Rlip-LNA using Lipofectamine 3000, and another set of cells was treated with 20 µg/well control IgG, Rlip-polyclonal antibody, or Rlip-monoclonal antibody. After incubation at 37 °C for 48 h, cell proliferation was assayed as described previously using MTT [58 (link)]. For colony-forming assays, 1 × 105 cells / 500 µL were incubated in 10 µg/mL R508 or scrambled-antisense for 24 h, and then aliquots of 50 or 100 µL were added to 60 mm Petri dishes containing 4 mL culture medium. After 10 days, adherent colonies were stained with methylene blue for 30 min and counted.
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