The importance of microglia for neurodegenerative diseases is well-known and these cells are therefore frequently used as a target for new pharmacological interventions.

Addition of Aβ1-42 coupled to pH-sensitive pHrodo™ Red label to isolated adult microglial cells of 5xFAD mice allows to monitor uptake and lysosomal degradation, measurable as increasing red fluorescence in the IncuCyte® Livecell imaging system.

Already 8 h after adding pHrodo™ Red labelled Aβ1-42, 5xFAD microglia show more than doubled fluorescence intensity compared to age-matched non-transgenic microglia, reflecting intense phagocytosis of Aβ in the 5xFAD microglia (Figure 1A). Although also non-transgenic microglia showed phagocytosis of Aβ1-42, this extremely strong phagocytotic response of 5xFAD microglia persisted with time and was at the end confirmed by measuring remaining Aβ1-42 in the supernatant of the cells. 5xFAD microglia took up significantly more Aβ1-42 compared to non-transgenic microglia observable as significantly reduced Aβ1-42 in the supernatant (Figure 1B).

Figure 1: Assessment of Aβ1-42 phagocytosis in isolated adult microglia of 5xFAD and non-transgenic (ntg) animals. Aβ1-42 phagocytosis was measured for 48 h using pHrodo™ Red labelled Aβ1-42 and IncuCyte® Livecell imaging (A). After 48 h of incubation, the supernatant of the same cells was analyzed for remaining Aβ1-42 (B). n=5 per group. Mean ± SEM. Two-tailed unpaired t-test; ***p<0.001.

Alternatively, also early postnatal primary microglia, the murine microglial cell line BV2 or iPSC-derived microglia can be used in the pHrodo™ Red phagocytosis assay for the assessment of Aβ1-42 uptake in response to compound treatment.

Furthermore, labelling of other disease-relevant peptides and proteins with pHrodo™ Red is possible.

Figure 2 Assessment of Aβ1-42 phagocytosis in BV2 cells using pHrodo™ Red labelled Aβ1-42 and IncuCyte® Livecell imaging. Representative images showing orange fluorescence in BV2 cells incubated with pHrodo™ Red labelled Aβ1-42 after 4h. Scale bar 200 µM.

Adult as well as early postnatal primary microglial cells of 5xFAD mice, the murine microglial cell line BV2 and iPSC-derived microglia are therefore well-suited to study phagocytosis in vitro.