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

Abstract

Source: Funes, S., et. al., Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes. J. Vis. Exp. (2022)

This video demonstrates an assay to study the phagocytic capacity of microglia-like cells using pH-sensitive fluorescent dye-labeled synaptosomes.

Protocol

1. Live-cell phagocytosis assay
   1. Plate 20-30 × 10⁴ PMPs into 96-well plates in 100 µL of iMG complete medium and follow the differentiation process for 10 days.
   2. On the day of the assay, prepare the nuclear staining solution by adding 1 drop of a nuclear stain for live cells into 2 mL of iMG basal medium.
   3. Remove 40 µL of medium per well of the 96-well plate and add 10 µL of the nuclear staining solution using a multichannel pipette. Incubate the plate at 37 °C and 5% CO₂ for 2 h.
   4. Thaw the labeled synaptosomes on ice and gently sonicate using a water sonicator for 1 min. Immediately transfer the synaptosomes back to ice. Dilute the labeled synaptosomes in iMG complete medium at a ratio of 1 µL of synaptosomes per 50 µL of the medium.
      NOTE: The concentration of synaptosome is assay-dependent and may require optimization. To maintain a relatively low percentage (i.e., 0.1% or less) of DMSO in the medium, it is advised not to add more than 2 µL of synaptosomes per well.
   5. As a negative control, pretreat some wells with cytochalasin D to inhibit actin polymerization and, thus, phagocytosis. Prepare a solution of 60 µM cytochalasin D in iMG complete medium. Add 10 µL of this solution to each well for a final concentration of 10 µM and incubate at 37 °C and 5% CO₂ for 30 min.
   6. Remove the plate from the incubator and incubate at 10 °C for 10 min. Maintain the plate on ice and add 50 µL of medium containing synaptosomes.
   7. Centrifuge the plate at 270 × g for 3 min at 10 °C and maintain the plate on ice until imaging acquisition.
2. Imaging acquisition and analysis
   1. Insert the plate into a live-cell imaging reader and select the wells to be analyzed.
   2. Select a 20x objective lens.
   3. Adjust the focus, light-emitting diode (LED) intensity, integration time, and gain of the brightfield and blue (4',6-diamidino-2-phenylindole [DAPI]) channels. The synaptosome fluorescence should be negligible at the initial time point. Use the brightfield channel as a reference to focus on the red (RFP) channel. The integration time and gain can vary between experiments; use these initial settings for the red channel: LED: 4, integration time: 250, and gain: 5.
   4. Select the number of individual tiles to be acquired in a montage per well (for example, acquire 16 tiles at the center of the well, thereby imaging approximately 5% of the total well area). Set the temperature to 37 °C and the desired time interval for imaging.
      NOTE: Images were acquired every 1 - 2 h for up to 16 h in this study.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cell culture materials
Primaria 96-well Clear Flat Bottom Microplate	Corning	353872
CellAdhere Laminin-521	STEMCELL Technology	77004	Referred as to laminin 521
Components of iMG base media
Advanced DMEM/F12	Gibco	12634010	Final concentration 1x
GlutaMAX	Gibco	35050061	Final concentration 1x
N2 supplement, 100x	Gibco	17502-048	Final concentration 1x
Penicillin-Streptomycin (10,000 U/mL)	Gibco	15140122	Final concentration 100 U/mL
Components of iMG complete media
55 mM 2-mercaptoethanol	Gibco	21985023	Final concentration 55 µM
IL-34	PeproTech or Biologend	200-34 or 577904	Final concentration 100 ng/mL
iMG base media			Final concentration 1x
M-CSF	PeproTech	300-25	Final concentration 5 ng/mL
TGF-β	PeproTech	100-21	Final concentration 50 ng/mL
Phagocytosis assay dyes
NucBlue Live Ready reagent	Invitrogen	R37605
pHrodo Red, succinimidyl ester	ThermoFisher Scientific	P36600	Referred as to pH-sensitive dye
Other cell-culture reagents
Cytochalasin D	Sigma		Final concentration 10 µM
Software and Equipment
Centrifuge	Eppendorf	Model 5810R
Cytation 5 live cell imaging reader	Biotek
Gen5 Microplate Reader and Imager Software	Biotek	version 3.03