Fitc dextran 4kd

For the phagocytosis assay 5 × 10⁴ iMG progenitors were seeded onto 8-well chamber slide (IB-80827, iBIDI) coated with PDL and matured for 7 days with 50% medium change every other day. Uptake assays were then performed by exchanging 100% media to fresh media supplemented with either 250 ηM Aβ_1–42 HyLite Fluor 647 (AS-60493, AnaSpec), mouse brain purified ^tdTomato-tagged synaptosomes generated in house, FITC-Dextran 4kD (46944, Sigma), FITC-Dextran 150 kD (46946, Sigma), Zymosan beads-568 (Z23374, ThermoFisher) and 75 nM LysoTracker DND-99 (L7528, ThermoFisher) for 100 min. After elapsed time, iMG were washed with PBS and fixed with 4% paraformaldehyde for 30 min. Fixed iMG were subsequently stained with SNL (FL-1301-2, Vector) to visualise the membrane and with 2 µM Hoechst (62249, ThermoFisher) nuclear stain. Images were acquired using Nikon A1R inverted confocal microscope (Nikon) running the NIS elements software using 60 × oil immersion lens. Acquired images were analysed using FIJI and IMARIS, thresholding to WT/Control, marking ROI and quantified either as % Cell Area or uptake (cargo) volume per total cell volume. Statistical significance was assessed using a Kruskal–Wallis test.

Mice fasting for 4 h were orally gavaged with 200 μL of FITC-dextran 4kD (440 mg kg⁻¹ of body weight) (Sigma, Germany), and blood was collected 4 h later. FITC-dextran serum concentration was measured by flow cytometry at 490 nm excitation wavelength and detected at 530 nm emission filter. Standard curve of serially diluted FITC-dextran was used for data fitting.