Developing Idelalisib-Resistant Cell Lines

VL51 cells were cultured according to the recommended conditions, as previously described.^{12 (link)} To develop resistance cell lines were exposed to a 90% inhibitory concentration (IC₉₀) of idelalisib (Selleckchem, Houston, TX, USA) for several months until they acquired specific drug resistance (resistant cells). In parallel, cells were cultured under similar conditions in the absence of drug (parental cells). Proliferation of stable resistance was tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay after 2 weeks of drug-free culture. The multidrug resistance phenotype was assessed by real-time polymerase chain reaction for MDR1 and MDR2/3 genes using published primers.^{13 (link)} We developed biological replicates by splitting the resistant clones 1 month after the development of resistance, keeping them separate for 6 months before performing further experiments. Cell line identity was periodically authenticated by short tandem repeat DNA profiling, as previously described.^{14 (link)} Cells were periodically tested to confirm Mycoplasma negativity using the MycoAlert My-coplasma Detection Kit (Lonza, Visp, Switzerland).