Astrocytes' response to Flunarizine combined with irradiation
Astrocytes' response to Flunarizine combined with irradiation
The cells used for the experiment are human primary astrocytes (3H Biomedical - SC1800). The cells received either DMSO or Flunarizine treatment at 10uM. 2 hours later, cells have been irradiated or not with 10Gy. 24 hours after irradiation the cells have been lysed and the RNA content extracted for bulk RNAseq analysis. 4 replicates have been made for each condition. Primary human astrocytes (3H Biomedical - SC1800-5) were cultured in astrocyte medium (3H Biomedical, SC1801) supplemented with 2% FBS, 1% PenStrep and astrocyte growth serum (provided by 3H Biomedical). one day after seeding, cells were treated with either DMSO or Flunarizine 10uM (MedChemExpress HY-B0358A) and 2 hours later irradiation was performed or not using a Cix1 x-ray irradiator (Xstrahl) at 10Gy. Cells were lysed 24 hours after irradiation The RNA content of the cells have been extracted RNeasy mini kit (Qiagen) together with the Qiashredder Kit (Qiagen) according to manufacturer's protocols Protocol: Illumina Stranded mRNA Prep, Ligation Reference Guide (Document # 1000000124518 v03). Modification of “Enrich DNA Fragment” step: 12 PCR cycles. Reagent/kit: Illumina® Stranded mRNA Prep, Ligation (20091659, Illumina) Indexing: Illumina® RNA UD Indexes Set C, Ligation (96 Indexes, 20091655, Illumina) Equipment: Automatisation of Clean up steps: King Fisher FLEX (18-5400620, ThermoScientific), incubations and PCR: Eppendorf Mastercycler X50s (6311000010, Eppendorf)