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…
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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 |
