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The changed cells were named EPT1.
The following day, medium was changed, cells were treated as indicated and harvested.
The medium was then changed, cells were maintained for 24 h, and then medium was collected to quantify ANGPTL2 protein by ELISA.
Thereafter, the culture medium was changed, cells were trypsinised and the number of EGFP-positive cells was controlled by flow cytometry, performed as described previously (Temme et al, 2003).
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Although both triterpenes displayed similar changes, cells were more sensitive to erythrodiol.
To test whether SFKs are involved in MCF-7 phenotypic changes, cells were treated with the SFK-specific inhibitor PP2.
To quantify the morphological changes, cells were stained and examined using automated fluorescence microscopy and image analysis software [ 18, 27].
At 48 h after the medium change, cells were selected in medium containing 400 μg ml−1 of G418 (GIBCO BRL) and G-418-resistant clones were established.
To exclude ex vivo neutrophil density changes, cells were separated within 2 hours of collection using Ficoll-Hypaque™ Plus (GE Healthcare Biosciences, Uppsala, Sweden) density gradient.
To evaluate possible morphological changes cells were treated with 1 μM of PDA-66 for 48 h and analyzed by light microscopy.
In response to extracellular changes, cells are well known to utilize the ubiquitin/proteasome system to control levels of transcription factors, cell-cycle proteins or membrane receptors.
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