Live-cell Phagocytosis Assay of Microglia-Like Cells Using Human Synaptosomes

Take a multi-well plate containing microglia-like cells exhibiting phagocytic activity.

Incubate with a nuclear stain to label nuclei.

Add an actin polymerization inhibitor to the negative control well.

During incubation, the inhibitor binds to actin filaments, inhibiting actin polymerization and phagocytosis.

Maintain the plate at low temperatures and add pH-sensitive dye-labeled human synaptosomes--isolated fragments of neural synaptic terminals.

Centrifuge the plate at low temperatures to promote cell-synaptosome interactions but prevent premature uptake.

Transfer the plate to a live-cell imaging system.

In the test well, synaptosomes are phagocytosed and fuse with lysosomes forming phagolysosomes. The lysosomal acidic environment activates the dye, resulting in its fluorescence.

Over time, in the test well, more synaptosomes are engulfed causing a further increase in fluorescence.

However, in the negative control well, actin polymerization is inhibited, preventing synaptosome uptake. The resulting minimal fluorescence confirms the fluorescence dependence on actin-mediated phagocytosis.

On the day of the assay, remove 40 microliters of medium per well, and add 10 microliters of the nuclear staining solution. Incubate the plate for two hours. Thaw the labeled synaptosomes on ice and gently sonicate using a water sonicator for one minute. Again, immediately place the synaptosomes on ice. Dilute the labeled synaptosomes and IMG complete medium at 1 microliter of synaptosomes per 50 microliters of medium.

For negative control, prepare 60 micromolar cytochalasin D in IMG complete medium to inhibit actin polymerization, and thus phagocytosis. Then, add 10 microliters of this solution to each well for a final concentration of 10 micromolar and incubate for 30 minutes. Remove the plate from the incubator and incubate at 10 degrees Celsius for 10 minutes. Maintain the plate on ice and add 50 microliters of medium containing synaptosomes.

Centrifuge the plate at 270 times g for three minutes at 10 degrees Celsius and maintain the plate on ice until imaging acquisition. Insert the plate into a live cell imaging reader and select the wells to be analyzed. Then, select a 20x objective lens. Next, adjust the focus Light Emitting Diode or LED intensity integration time and gain of the bright field and blue channels. The synaptosomes fluorescence should be negligible at the initial time point.

Select the number of individual tiles to be acquired in a montage per well. Acquire 16 tiles at the center of the well, imaging approximately 5% of the total well area. Set the temperature to 37 degrees Celsius and the desired time interval for imaging.