Mycoalert detection kit

KL-ROS1 is a cell line that we established in vitro from a lung carcinoma that arose spontaneously in a 40-week-old K-Ras^G12D female mouse. Cells are cultured in Dulbecco Modified Eagle’s Medium (DMEM) supplemented with 20% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich) and routinely checked for Mycoplasma contamination using the Mycoalert Detection Kit (Lonza, Zurich, Switzerland) and consistently found to be negative. In order to test ROS1 expression, KL-ROS1 cells were washed in PBS supplemented with 0.2% bovine serum albumin (BSA) and 0.01% sodium azide (Sigma-Aldrich) and stained for ROS1 after fixation and permeabilization with the Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences, Allschwil, Switzerland). In order to detect the ROS1 positivity, a primary rabbit anti-ROS1 (D4D6) mAb (Cell Signaling Technology) and a secondary fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobulins (Dako, Santa Clara, CA, USA) were used. For transplantable tumor experiments, wt mice were challenged subcutaneously with different doses of KL-ROS1 cells, ranging from 10⁵ to 10⁶. The tumor dimensions of the challenged mice were inspected weekly by palpation. Progressively growing masses >1 mm in diameter were regarded as tumors. Mice were sacrificed when the tumor exceeded 10 mm in diameter.

Human breast cancer cell lines, MCF-7 and T47D were purchased from ATCC and were cultured in phenol red–free DMEM (Gibco) with 10% FBS, 0.1% insulin, 50 U/mL penicillin/streptomycin, 1% nonessential amino acids (Gibco). Tamoxifen resistant (TamR) MCF-7 and T47D cells were generated previously^{32 (link)}. Tamoxifen and GebR-7b were purchased from Sigma. Liproxstatin-1 and RSL3 were purchased from MedChem Express. Cells were routinely tested for mycoplasma contamination using MycoAlert detection kit (Lonza) and were authenticated by STR sequencing.