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Spheroid formation assay

NB Narumol Bhummaphan
PP Piyapat Pin-on
PP Preeyaporn Plaimee Phiboonchaiyanan
JS Jirattha Siriluksana
CA Chatchawit Aporntewan
PC Pithi Chanvorachote
AM Apiwat Mutirangura
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The formation of spheroids was performed under serum-free conditions in an ultralow attachment plate as previously reported [7]. NSCLC-derived H292 and A549 cells were pretreated with PNA-A15 and scramble (5 μM) for 48 h. Then, cells were detached using 1 mM EDTA and suspended into single cells. These cells were grown in a 24-well ultralow attachment plate at a density of 2.5 × 103 cells/well in stem cell media (SCM) (i.e., 0.8% (w/v) methylcellulose-based serum-free medium (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 20 ng/ml epidermal growth factor (BD Biosciences, San Jose, CA, USA), 20 ng/ml basic fibroblast growth factor and 4 mg/ml insulin (Sigma) for 7 days to form primary spheroids. These primary spheroids were harvested, resuspended as single cells using 1 mM EDTA and cultured in SCM for 14 days in a 24-well ultralow attachment plate to form secondary spheroids.

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