Microglial Phagocytosis Assay: An In Vitro Assay to Establish Microglial Phagocytosis Model for Neuroblastoma Cells Using iPSC Macrophages

Microglia are brain macrophages that help remove pathogens, cell debris, and cancerous nerve cells or neuroblastoma by a process called phagocytosis.

To mimic phagocytosis in vitro, begin with a culture of neuroblastoma cells fixed in paraformaldehyde.

Paraformaldehyde, a chemical fixative, causes random distribution of membrane phospholipids like phosphatidylserine, resulting in their translocation to the cell membrane's outer surface while simultaneously stabilizing the cell structure.

Now, add a pH-sensitive amine-reactive fluorescent dye to the neuroblastoma cell culture and incubate.

The dye molecules bind to the amine groups of phosphatidylserine on the neuroblastoma cell surface.

Centrifuge the tube and remove the supernatant containing unbound dye molecules. Rinse the cell pellet with a suitable buffer and centrifuge.

Remove the supernatant and resuspend the pellet in the culture medium.

Next, seed pre-stained macrophages in a culture plate. Stained macrophages will appear red with a blue nucleus.

Add stained neuroblastoma cells to this culture and incubate.

The macrophage-phagocytic receptors recognize the exposed phosphatidylserine on neuroblastoma cells and internalize them in membrane compartments called phagosomes.

The phagosomes migrate and fuse with lysosomes, exposing neuroblastoma cells to the lysosome's low pH environment. Low pH activates the dye molecules on neuroblastoma cells, causing the dye color to change.

Under a fluorescence microscope, the phagocytosed neuroblastoma cell appears yellow inside the red macrophages.

Pipette the SH-SY5Ys into a 15-milliliter conical centrifuge tube. Centrifuge at 400 x g for 5 minutes. Aspirate the supernatant and resuspend the cells in 2 milliliters of phenol red-free HEPES-buffered media, making sure to break up clumps before fixation.

Fix the cells by adding 2 milliliters of 4% paraformaldehyde to the tube and incubating for 10 minutes at room temperature with occasional gentle agitation. Add 10 milliliters of HBSS to the tube.

Then, centrifuge at 1,200 x g for 7 minutes. Aspirate the supernatant and resuspend the cell pellet in 2 milliliters of phenol red-free HEPES-buffered media. Count and remove one million SH-SY5Y cells into a 2-milliliter low protein binding tube.

Bring the total volume to 300 to 500 microliters with phenol red-free HEPES-buffered media. Then, briefly warm the tube in the 37 degrees Celsius water bath. Reconstitute the pH-sensitive red fluorescent dye STP ester according to manufacturer's instructions.

Then, add 12.5 micrograms of dye per million SH-SY5Y cells. Mix gently by flicking the tube and incubate the tube at room temperature for 30 minutes protected from light.

Add 1 milliliter of HBSS and centrifuge at 1,200 x g for 7 minutes and 4 degrees Celsius. Discard the supernatant and wash with 2 milliliters of HBSS.

Resuspend the cell pellet in phenol red-free macrophage media to a concentration of 0.2 to 1.2 million cells per milliliter so that 50 microliters contain 10,000 to 60,000 cells.

In a biological safety cabinet, prepare a solution of deep red fluorescent, cell-permeant, succinimidyl ester-reactive dye in macrophage media. Add Hoechst 33342 and warm the working solution to 37 degrees Celsius in a water bath.

Aspirate the iPSC macrophage medium gently by pipetting the cell supernatant with a multichannel pipette into a sterile reservoir.

Add 70 microliters per well of the dye solution to the iPSC macrophages using a multichannel pipette. Incubate for 1 hour at 37 degrees Celsius and 5% carbon dioxide.

Prepare experimental treatments in phenol red-free macrophage media. After the incubation, aspirate the iPSC macrophage medium very gently with a multichannel pipette and add 100 microliters of HBSS per well.

Immediately remove HBSS by gentle pipetting. Then, add 100 microliters of phenol red-free macrophage media plus experimental treatments in separate wells. Incubate for 10 minutes to 1 hour at 37 degrees Celsius and 5% carbon dioxide.

Use a multichannel pipette to add 50 microliters of the labeled SH-SY5Ys per well from the sides of each well at the edge of the liquid. Then, incubate at 37 degrees Celsius and 5% carbon dioxide for 3 to 5 hours.

After the phagocytosis incubation, gently aspirate cell supernatants by pipetting with the multichannel pipette and discard. Wash once with 100 microliters of PBS.

Then, fix the cells by adding 100 microliters of 2% paraformaldehyde and incubating for 15 minutes at room temperature. Aspirate wells and add 100 microliters of PBS.

Either proceed directly to imaging with the high-content microscope or cover with plate sealer and foil and store the plate at 4 degrees Celsius until required.