SUMMARY
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- GPR32 knockout in microglia should worsen neuroinflammation if this is the primary mechanism
- Dose-response studies showing therapeutic window
## Background and Rationale
This falsification study rigorously tests the hypothesis that GPR32 serves as a protective receptor in microglial-mediated neuroinflammation by examining the consequences of receptor loss in controlled cellular models. The experiment employs CRISPR-Cas9 gene editing to generate GPR32 knockout BV2 microglia cell lines,
METHODOLOGY NOTES
**Phase 1: Cell Line Preparation and Genetic Modification (Days 1-14)**
• Generate GPR32 knockout BV2 microglial cell lines using CRISPR-Cas9 system with dual guide RNAs targeting exons 2 and 3
• Validate knockout efficiency by Western blot, qPCR, and sequencing (n=3 independent clones)
• Maintain wild-type BV2 controls and establish stable cell cultures in DMEM + 10% FBS
• Prepare immortalized human microglial cell line (HMC3) as secondary validation model
**Phase 2: Neuroinflammation Induction Protocol (Days 15-16)**
• Treat cells with LPS (0.1, 1.0, 10 μg/mL) + IFN-γ (20 ng/mL) for 24h to induce neuroinflammation
• Include ATP (5 mM, 30 min) treatment for NLRP3 inflammasome activation
• Establish co-culture system with primary neurons (DIV 7-10) to assess neuronal damage
• Include vehicle controls and unstimulated baseline conditions
**Phase 3: Dose-Response Analysis (Days 17-21)**
• Test GPR32 agonist RvD1 at concentrations: 0.1, 1, 10, 100 nM, 1 μM over 4h and 24h timepoints
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