Phase 1: Primary Cell Culture and SPP1 Manipulation — Week 1-2
Isolate primary microglia from P0-P2 C57BL/6J pups using magnetic-activated cell sorting (MACS) with CD11b+ microbeads (Miltenyi Biotec, 130-049-601). Maintain in DMEM/F12 with 10% FBS, 1% penicillin/streptomycin, and 10 ng/mL M-CSF (R&D Systems, 416-ML-010). Transfect microglia (n=6 wells per condition) with SPP1 siRNA (50 nM, Dharmacon L-040777-01-0005) or scrambled control using Lipofectamine RNAiMAX (Thermo Fisher, 13778150). Confirm 70-80% knockdown by qRT-PCR after 48h. Prepare amyloid-β1-42 oligomers (Anaspec, AS-24224) at 5 μM by dissolving in DMSO, diluting in phenol-red-free F12, and incubating at 4°C for 24h.
...Phase 1: Primary Cell Culture and SPP1 Manipulation — Week 1-2
Isolate primary microglia from P0-P2 C57BL/6J pups using magnetic-activated cell sorting (MACS) with CD11b+ microbeads (Miltenyi Biotec, 130-049-601). Maintain in DMEM/F12 with 10% FBS, 1% penicillin/streptomycin, and 10 ng/mL M-CSF (R&D Systems, 416-ML-010). Transfect microglia (n=6 wells per condition) with SPP1 siRNA (50 nM, Dharmacon L-040777-01-0005) or scrambled control using Lipofectamine RNAiMAX (Thermo Fisher, 13778150). Confirm 70-80% knockdown by qRT-PCR after 48h. Prepare amyloid-β1-42 oligomers (Anaspec, AS-24224) at 5 μM by dissolving in DMSO, diluting in phenol-red-free F12, and incubating at 4°C for 24h.
Phase 2: Synaptosome Preparation and Co-culture Setup — Week 2
Prepare synaptosomes from adult mouse cortex using Percoll gradient centrifugation. Homogenize tissue in sucrose buffer (0.32M sucrose, 1mM EDTA, 5mM HEPES pH 7.4), centrifuge at 1,000g for 10min, then layer supernatant on Percoll gradients (3%, 10%, 23% in sucrose buffer). Collect synaptosome fraction at 10-23% interface. Label synaptosomes with pHrodo Red (Thermo Fisher, P36600) at 10 μg/mL for 30min at 37°C. Wash and resuspend at 1×10^6 synaptosomes/mL. Add labeled synaptosomes to microglial cultures (1:5 ratio) along with Aβ oligomers (5 μM) or vehicle control.
Phase 3: Real-time Phagocytosis Monitoring — Week 3
Monitor synaptic engulfment using live-cell confocal microscopy (Zeiss LSM 880) with 63x objective. Image every 10 minutes for 4 hours at 37°C with 5% CO2. Quantify pHrodo Red signal intensity within CD11b+ microglia using ImageJ macros. Calculate phagocytic index as (number of pHrodo+ particles per microglia × percentage of phagocytic microglia)/100. Include controls: vehicle-treated synaptosomes, cytochalasin D treatment (10 μM, Sigma C8273) to block phagocytosis, and heat-inactivated synaptosomes.
Phase 4: Molecular Marker Analysis — Week 3-4
Harvest microglia at 4, 8, and 24h post-synaptosome exposure for qRT-PCR and Western blot analysis. Extract RNA using RNeasy Mini Kit (Qiagen, 74104) and synthesize cDNA with SuperScript III (Thermo Fisher, 18080051). Quantify mRNA expression of phagocytic markers: C1qa (primers: F-GGCTACCAGTCCTCAACCTG, R-TTGCTGTCCCAGTCTCAAGG), Grn (F-TGTGGATGTGGAGCAAGATG, R-CTGCAGGAGGTGGAGTTCAT), Ctsb (F-CCAGTCTAACGGGCACAAAT, R-TTTGGTCATGGCCATCAGTG), and SPP1 (F-CCAAGTAAGTCCAACGAAAG, R-GGTGATGTCCTCGTCTGTA). Normalize to Gapdh. Perform Western blots for C1qA (Abcam ab182451, 1:1000), GRN (Abcam ab108608, 1:1000), and CTSB (Cell Signaling 31718, 1:1000) proteins.
Phase 5: Flow Cytometry Validation — Week 4
Analyze microglial activation and phagocytic capacity by flow cytometry. Fix cells in 4% PFA, permeabilize with 0.1% Triton X-100, and stain with primary antibodies: CD11b-PE (BD Biosciences 553311, 1:200), CD68-FITC (BioLegend 137008, 1:100), and intracellular C1qA-APC (custom conjugated ab182451, 1:100). Include isotype controls and single-stain compensation controls. Acquire 10,000 events per sample on BD FACSCanto II. Gate on viable CD11b+ cells and analyze CD68 and C1qA expression levels.
Phase 6: Statistical Analysis and Validation — Week 4-5
Perform statistical analysis using GraphPad Prism 9. Use two-way ANOVA with Tukey's post-hoc test for multiple comparisons (treatment × time). Calculate sample size requirements for 80% power to detect 40% difference with α=0.05. Validate key findings using independent microglial cultures from APP/PS1 mice (JAX 005864) and repeat critical experiments with recombinant SPP1 protein (R&D Systems 441-OP-050) at 100 ng/mL.