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

Published: April 30, 2023

Abstract

Source: Hall-Roberts, H. et al. In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages. J. Vis. Exp. (2021)

In this video, we perform the microglial phagocytosis assay for neuroblastoma using induced pluripotent stem cells. The assay helps assess the impairment of microglial function in neurodegenerative diseases.

Protocol

1. Labeling of Dead SH-SY5Ys with pH-sensitive Red Fluorescent Dye 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. Reconstitute the pH-sensitive red fluorescent dye…

開示

The authors have nothing to disclose.

Materials

15 mL conical centrifuge tube Falcon 352096 For centrifugation of cells
2-20 µL, 20-200 µL, 100-1000 µL single-channel micropipettes
4% paraformaldehyde in PBS Alfa Aesar J61899 For fixation of cells
6-well plate, tissue culture treated
AggreWell-800 24-well plate STEMCELL Technologies 34815 Microwell low-adherence 24-well plate for formation of embryoid bodies
Automated cell counter
Benchtop centrifuge
Benchtop microcentrifuge
CellCarrier-96 Ultra Microplates, tissue culture treated, black, 96-well with lid Perkin Elmer 6055302 96-well tissue culture (TC)-treated microplate with black well walls and an optically-clear bottom, for phagocytosis assay
CellProfiler software Open-source software for analysis of phagocytosis images obtained by live-cell time-lapse microscope. Download for free from website (http://cellprofiler.org/), this protocol used version 2.2.0.
CellTracker Deep Red dye Thermo Fisher C34565 Deep red-fluorescent, cell-permeant, succinimidyl ester-reactive dye for staining cytoplasm of iPS-macrophages. Dissolve CellTracker Deep Red dye in DMSO to 2 mM (1.4 mg/mL). Use at 1 μM, by dilution of DMSO stock with Macrophage media. 
Class 2 laminar air flow safety cabinet
CO2 gas bottle Accessory for EVOS FL Auto
CO2 incubator, set to 37°C and 5 % CO2
Cytochalasin D Cayman 11330 Negative control treatment for phagocytosis assay. Reconstitute in DMSO to 10 mM and store aliquots at -20°C, avoid further freeze-thaw cycles. Use at final concentration 10 µM.
DMEM/F12 Gibco 11320074 Component of SH-SY5Y media
DMSO Sigma D8418 Solvent for CellTracker and pHrodo dyes
Fetal Bovine Serum Sigma F4135 Component of SH-SY5Y media
Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix Invitrogen A1413302 hESC-qualified basement membrane matrix for iPSC culture
GlutaMAX Supplement Gibco 35050-038 Component of both Factory and Macrophage media
HBSS Lonza BE 10-547F Hank’s balanced salt solution for washing steps
Human recombinant IL-3 Gibco PHC0033 Component of both Factory and Macrophage media
Live Cell Imaging Solution Thermo Fisher A14291DJ Phenol red-free HEPES-buffered media for labelling dead SH-SY5Ys
Low protein binding 2 mL tubes Eppendorf 30108.132 For staining SH-SY5Ys
M-CSF Thermo Fisher PHC9501 Component of both Factory and Macrophage media
mTeSR1 Medium STEMCELL Technologies 85850 iPSC media
Multichannel 20-200 uL pipette For liquid handling of 96-well plate
NucBlue Live ReadyProbes Reagent Thermo Fisher R37605 Hoechst 33342 formulation in a dropper bottle for staining nuclei of iPS-macrophages, use 0.5 drops/mL in Macrophage media.
Opera Phenix High-Content Screening System Perkin Elmer HH14000000 High-content imaging microscope, used with Harmony software version 4.9.
Penicillin-Streptomycin Gibco 15140-122 Component of Factory, Macrophage, and SH-SY5Y media
pHrodo iFL Red STP-Ester Thermo Fisher P36011 pH-sensitive red fluorescent dye for labelling dead SH-SY5Ys. Reconstitute pHrodo iFL Red STP Ester powder in DMSO to a 5 mg/mL concentration. For each 1 million SH-SY5Ys, add 2.5 μL (12.5 μg) of pHrodo iFL Red STP Ester stock to pre-warmed cells suspended in Live Cell Imaging Solution. 
Serological pipette filler
T175 flask, tissue culture treated Vessel for differentiations of iPSC-macrophage precursors, known as "Factories"
T75 flask Vessel for SH-SY5Y culture
Transparent plate sealers Greiner Bio-One 676001 For assay plate storage and transportation
Water bath, set to 37°C
X-VIVO 15 Medium with L-glutamine, gentamicin, and phenol red Lonza BE04-418F Component of Factory and Macrophage media
X-VIVO 15 Medium with L-glutamine; without gentamicin or phenol red Lonza 04-744Q Phenol red-free macrophage media, for use in phagocytosis without additives or growth factors

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記事を引用
Microglial Phagocytosis Assay: An In Vitro Assay to Establish Microglial Phagocytosis Model for Neuroblastoma Cells Using iPSC Macrophages. J. Vis. Exp. (Pending Publication), e20615, doi: (2023).

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