Protocol for qualitative analysis of lysosome immunoprecipitation from patient-derived glioblastoma stem-like cells

Lysosomes are critical for the sustenance of glioblastoma stem-like cells (GSCs) properties. We present a protocol to enrich and purify lysosomes from patient-derived GSCs in culture. We describe the steps required to stably express a tagged lysosomal protein in GSCs, mechanically lyse cells, magnetically immunopurify lysosomes, and qualitatively assess these organelles. We then detail the procedure for retrieving intact and purified lysosomes from GSCs. We also specify cell culture conditions, storage procedures, and sample preparation for immunoblotting. For complete details on the use and execution of this protocol, please refer to Maghe et al.1 [Display omitted] •Generating patient-derived glioblastoma cells expressing lysosomal tagged proteins•Retrieving intact organelles from mechanical cell lysis•Isolating intact lysosomes with magnetic bead immunoprecipitation•Evaluating the qualitative features of purified lysosomes with western blots Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Lysosomes are critical for the sustenance of glioblastoma stem-like cells (GSCs) properties. We present a protocol to enrich and purify lysosomes from patient-derived GSCs in culture. We describe the steps required to stably express a tagged lysosomal protein in GSCs, mechanically lyse cells, magnetically immunopurify lysosomes, and qualitatively assess these organelles. We then detail the procedure for retrieving intact and purified lysosomes from GSCs. We also specify cell culture conditions, storage procedures, and sample preparation for immunoblotting.