HCC cells were cultured in DMEM (11965092, Thermo Fisher Scientific) containing 10% FBS in an incubator containing 5% CO2 at 37°C. When cell confluence reached 80%, the cells were resuspended in a stem-cell-conditioned medium. Next, the cells were plated in six-well cell culture plates with ultralow adhesion and cultured for 6 days in an incubator containing 5% CO2 at 37°C. When the tumorspheres grew to 50 μm, the cell suspension was collected and centrifuged at 600 rpm for 5 min. In the next step, the tumorspheres were detached with 0.25% trypsin/0.02% EDTA in a 37°C incubator for 2 min, dissociated into single cells, and then centrifuged at 1000 rpm in 5 mL of PBS at 18°C for 5 min before the supernatant was discarded. The cells were resuspended in the stem-cell-conditioned medium. The cells were then counted and the experiment was conducted three times to obtain the average values. The cells were then plated in a six-well cell culture plate with ultralow adhesion (ultralow plates, Becton Dickinson, NJ, USA) in an incubator containing 5% CO2 at 37°C.
Ultralow plates
Manufactured by BD
Sourced in United States
Ultralow plates are a type of laboratory equipment designed for various applications that require extremely low temperatures. These plates are capable of maintaining and regulating temperatures down to ultra-low levels, typically below -80°C. They are often used in cryogenic storage and research applications that necessitate the preservation of samples or materials at very low temperatures.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using ultralow plates
1
Culturing and Maintaining Tumorspheres
2
Serum-free 3D Culture of Pancreatic Cancer Cells
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A standard cell culture medium (Gibco, Carlsbad, CA, USA) consisted of serum-free DMEM with addition of hepatocyte growth factor (10 ng/mL), basic fibroblast growth factor (bFGF) (10 ng/mL), and Noggin (10 ng/mL) + LIF (1000 U/mL). PANC-1 cells or MIA PaCa-2 cells were seeded in 96-well plate (ultralow plates, Becton, Dickinson and Company, NJ, USA), with 2 mL DMEM containing 10% FBS at 37 °C with 5% CO2. Half culture medium was replaced by fresh culture medium every 2 day and the subculture was carried out once the cell sphere diameter was larger than 50 μm. The adhered cells were abandoned during the subculture and the suspended cells were collected by centrifugation at 300 × g for 3 min. After removing the supernatant, cells were treated with 0.25% trypsin and 0.02% ethylene glycol-bis (β-aminoethyl ether)-N, N-tetraacetic acid. The cells were subsequently gently triturated with fresh medium into single-cell suspension. After being centrifuged at 300 × g for 3 min, the supernatant was discarded, and the collected cells were cultured with fresh culture medium. After counting, the cells were seeded to a new six-well plate at a density of 1 × 104 cell/well and the subculture was kept for 2 weeks continuously.
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