Proposed experiment from debate on Senolytics targeting p16/p21+ senescent astrocytes and microglia may reduce SASP

SUMMARY

# Proposed experiment from debate on Senolytics targeting p16/p21+ senescent astrocytes and microglia may reduce SASP ## Background and Rationale This falsification study investigates whether senescent astrocytes and microglia directly transfer toxic lipid peroxidation products to neurons, contributing to neurodegeneration through senescence-associated secretory phenotype (SASP). The experiment utilizes real-time tracking of fluorescently-labeled lipid peroxidation products in co-culture systems

METHODOLOGY NOTES

**Phase 1: Cell Culture Preparation (Days 1-7)** • Establish primary astrocyte and microglia cultures from C57BL/6 mice (n=6 biological replicates) • Induce cellular senescence using 10 Gy ionizing radiation or 100 μM H₂O₂ treatment • Confirm senescence markers: p16^INK4a^, p21^CIP1^, SA-β-gal activity, and SASP cytokine secretion (IL-6, TNF-α) • Establish co-cultures with primary neurons (1:1:2 ratio astrocyte:microglia:neuron) • Validate senescent cell populations by flow cytometry (>80% p16⁺/p21⁺) **Phase 2: Fluorescent Lipid Peroxidation Tracking (Days 8-14)** • Load senescent cells with BODIPY C11 (10 μM) and C11-BODIPY 581/591 for real-time lipid peroxidation detection • Use confocal live-cell microscopy with 30-second intervals over 48-hour periods • Track transfer of oxidized lipid products using photobleaching recovery and particle tracking algorithms • Quantify colocalization coefficients between lipid peroxidation signals and neuronal compartments • Measure fluorescence int