Facs calibur cell cytometer

Cell cycle analysis was performed on cells treated for 24 hours, or left untreated, with increasing concentrations of crizotinib or TAE684 inhibitors. The cells were washed in ice-cold PBS, fixed in cold 70% ethanol and resuspended in 0.6 ml PI [50 μg/ml] right before cell cycle analysis with BD FACS-Calibur Cell Cytometer and Macintosh CellQuest software (Becton Dickinson, Italy).
Cell viability was measured by MTT assay every 24 hours up to three days. RMS, NB and ALCL cells were seeded onto 96-well microculture plates 12 hours before drug addition and then grown in the presence or absence of ALK inhibitors as indicated in the text. MTT optical density was measured with Victor3 Multilabel Counter spectrophotometer at 540 nm and values of three independent experiments averaged. Drug-induced growth inhibition of RMS cells in the presence or absence of HGF, IGF-I or EGF growth factors was measured alike.

To assess ALK expression at the plasma membrane, exponentially growing cells were incubated with 10 μg/ml anti-ALK primary monoclonal antibody, washed in PBS, and subsequently labeled with Alexa Fluor 488 conjugated anti-mouse secondary antibody. Staining with primary and fluorochrome-labeled secondary antibodies was performed on ice with ice-cold reagents/solutions, since low temperatures prevent the modulation and internalization of surface antigens that can produce a loss of fluorescence intensity. The cells were kept in the dark, in ice, until fluorescence analysis with BD FACS-Calibur Cell Cytometer (Becton Dickinson, Italy) was carried out. ALK mean fluorescence intensity (MFI) was measured by Macintosh CellQuest software (Becton Dickinson, Italy).