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

1. Labeling of Dead SH-SY5Ys with pH-sensitive Red Fluorescent Dye

1. Begin with fixed SH-SY5Y cells suspended in phenol red-free HEPES buffered media. Count the cells and remove the total number of cells required into a 2 mL low protein-binding tube. For each 1 million SH-SY5Ys, make up the total volume in the 2 mL tube to 300–500 µL with phenol red-free HEPES-buffered media. Warm the tube briefly in a 37 °C water bath.
2. Reconstitute the pH-sensitive red fluorescent dye STP ester (see Table of Materials) and add 12.5 µg of dye per million SH-SY5Y to the warm 2 mL tube of cells. Mix gently by pipetting. Incubate the tube at room temperature for 30 min, protected from light.    
   NOTE: The STP ester species of the pH-sensitive dye reacts with primary amines, and therefore, the labeling buffer must not contain free amines. Due to potentially limited solubility in aqueous buffers, add the DMSO-dissolved dye only to warm the aqueous buffer, mix immediately, and examine for signs of precipitate (dark particles under a light microscope).
3. Add 1 mL of HBSS and centrifuge at 1200 x g for 7 min at 4 °C. Discard the supernatant and wash with 2 mL HBSS. Repeat the centrifugation.
4. Discard the supernatant and re-suspend the cells in phenol red-free macrophage media (see Table of Materials) to a concentration of 200,000–1.2 million cells/mL so that 50 µL is 10,000–60,000 cells (i.e., 0.5x–3x more SH-SY5Ys than iPSC-macrophages).
   NOTE: Phenol red in media increases background fluorescence, and therefore, phenol red-free media should be used if live-cell imaging is to be performed. Storage of the stained SH-SY5Ys for more than a few hours is not recommended as this has not been assessed. Keep stained SH-SY5Ys on ice and protect from light.

2. Staining of iPSC-macrophages

1. In a biological safety cabinet, prepare a solution in macrophage media of a deep red-fluorescent, cell-permeant, succinimidyl ester-reactive dye (see Table of Materials). Add Hoechst 33342 (see Table of Materials). Warm the working solution to 37 °C in a water bath.
2. Aspirate the iPSC-macrophage medium gently by pipetting the cell supernatant with a multichannel pipette into a sterile reservoir. Add 70 µL/well of the dye solution prepared in step 2.1 to iPSC-macrophages, using a multichannel pipette. Incubate for 1 h at 37 °C and 5% CO₂.
3. Prepare experimental treatments in phenol red-free macrophage media. Include 10 µM cytochalasin D as a negative control treatment. Post-incubation, aspirate the iPSC-macrophage medium very gently with a multichannel pipette and add 100 µL/well of Hank's buffered saline solution (HBSS), to wash. Immediately remove HBSS by gentle pipetting, then add 100 µL of media ± compounds. Incubate for 10 min–1 h at 37 °C and 5% CO₂.
   NOTE: Cytochalasin D is a potent actin inhibitor and blocks phagocytosis. For any experimental treatments that require longer incubation, e.g., 24–72 h, perform the experimental treatment before step 2.1, using 100 µL/well of treatment in full macrophage media. Follow steps 2.1–2.3 as per the protocol so that cell staining is performed and subsequently the treatment is reapplied in phenol red-free macrophage media for the remainder of the phagocytosis assay.

3. Imaging Phagocytosis

1. Fixed-cell high-content imaging
   1. Use a multichannel pipette to add 50 µL of the labeled SH-SY5Ys per well, adding to the side of each well at the edge of the liquid. Incubate at 37 °C and 5% CO₂ for 3-5 h.
   2. After the phagocytosis incubation, gently aspirate cell supernatants by pipetting with a multichannel pipette, and discard. Wash once with 100 µL PBS.
   3. Fix the plate by addition of 100 µL of 2% paraformaldehyde and incubate for 15 min at room temperature.
   4. Aspirate wells and add 100 µL of PBS. Cover with plate sealer and foil; store at 4 °C until required.       
      NOTE: The assay plate can be stored like this for at least a week without significant signal degradation; longer storage has not been tested.
   5. Turn on the high-content imaging microscope (see Table of Materials) and open the image capture software. Load the assay plate into the microscope by clicking on the Load icon at the top of the screen.
   6. Select the Setup tab. In drop-down menus of the top left box: select the appropriate plate type, select the autofocus option Two Peak (Default), select the objective 40x Water, NA1.1, select Confocal mode, and select binning of 1.
   7. Flush the 40x water objective before use via the Settings menu.
   8. In the Channel Selection box, use the + icon to add the channels DAPI, Alexa 647, and Alexa 568. Set these to measure at a single plane of 1 µm. Optimize Time and Power settings for the staining efficiency of the assay plate.
      NOTE: As a guideline, set DAPI at 200 ms exposure and 100% power, Alexa 647 at 1500 ms exposure and 100% power, and Alexa 568 at 100 ms exposure and 40% power.
   9. Ensure the channels are not measured simultaneously by clicking on Channel Sequence to separate out the channels.
   10. Under Navigation | Define Layout, select the wells of measurement and select 9–12 fields per well.
   11. During set up, click on a representative field on the plate map, and check each measurement channel in turn, to ensure the staining is present and that the images are focused, by adjusting the channel offset.
   12. For data to be uploaded to a server for remote analysis, click on the Online Jobs box and the relevant screen name; this will enable automatic uploading of the data to a server after imaging.
   13. Save the assay protocol by clicking on the Save button.
   14. Click on the Run Experiment tab at the top and name the experiment plate, then click on Start.