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The experiment employs CRISPR-Cas9 gene editing to generate GPR32 knockout BV2 microglia cell lines,","entities":{"genes":["GPR32"],"diseases":["Neuroinflammation"]},"model_system":"cell_line","_schema_version":1,"experiment_type":"falsification","primary_outcome":"Measurement of pro-inflammatory cytokine release (IL-1β, TNF-α) in GPR32 knockout versus wild-type BV2 microglia following LPS stimulation, with the hypothesis predicting significantly increased inflammation in knockout cells.","methodology_notes":"**Phase 1: Cell Line Preparation and Genetic Modification (Days 1-14)**\n• Generate GPR32 knockout BV2 microglial cell lines using CRISPR-Cas9 system with dual guide RNAs targeting exons 2 and 3\n• Validate knockout efficiency by Western blot, qPCR, and sequencing (n=3 independent clones)\n• Maintain wild-type BV2 controls and establish stable cell cultures in DMEM + 10% FBS\n• Prepare immortalized human microglial cell line (HMC3) as secondary validation model\n\n**Phase 2: Neuroinflammation Induction Protocol (Days 15-16)**\n• Treat cells with LPS (0.1, 1.0, 10 μg/mL) + IFN-γ (20 ng/mL) for 24h to induce neuroinflammation\n• Include ATP (5 mM, 30 min) treatment for NLRP3 inflammasome activation\n• Establish co-culture system with primary neurons (DIV 7-10) to assess neuronal damage\n• Include vehicle controls and unstimulated baseline conditions\n\n**Phase 3: Dose-Response Analysis (Days 17-21)**\n• Test GPR32 agonist RvD1 at concentrations: 0.1, 1, 10, 100 nM, 1 μM over 4h and 24h timepoints\n• A","replication_status":"single_study","extraction_metadata":{"backfill_at":"2026-04-16T01:00:16.908595","needs_review":true,"extraction_notes":"Backfilled from debate_extraction source (no PMID 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