Synaptosomes45 and crude CNS myelin46 were purified as described previously, and conjugated with pHrodo™ Red, succinimidyl ester (Thermo Fisher Scientific, P36600) in 0.1 M sodium carbonate (pH 9.0) at room temperature with gentle agitation. After two-hour incubation, unbounded pHrodo was washed-out by multiple rounds of centrifugation and pHrodo-conjugated synaptosomes/myelin were re-suspended with isotonic buffer containing 5% DMSO for subsequent freezing. Purified control and A1 reactive astrocytes from P6 rat pups (see above) were incubated with 5 μl pHrodo-conjugated synaptosomes for 24 h, or 800 μg/ml media pHrodo-conjugated myelin debris and imaged at 1 h intervals. Live astrocytes were imaged with epifluorescence time lapse microscope (IncuCyte Zoom ® System) to reveal engulfed pHrodo-conjugated particles. For image processing analysis, we took 9 images/well using 20× objective lens from random areas of the 24 well plates and calculated the phagocytic index (PI) by measuring the area of engulfed synaptosomes/myelin (fluorescent signal) normalized to the area of astrocytes, using ImageJ. Relative engulfment ability was calculated by normalizing the PI of control (non-reactive) astrocytes by that of A1 reactive astrocytes3.
