Apr 19, 2026
Induced Microglia differentiation from iPSC
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: Elena Coccia 2026. Induced Microglia differentiation from iPSC. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwn2yzvmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 17, 2026
Last Modified: April 19, 2026
Protocol Integer ID: 243538
Keywords: AggreWell, embryoid bodies, iPSCs, cell culture, differentiation, induced microglia differentiation from ipsc, induced microglia differentiation, seeding ipsc, generating embryoid body, embryoid body, preparing aggrewell plate, ipsc, aggrewell plate
Disclaimer
This protocol was generated by AI
Abstract
This protocol outlines the steps for preparing AggreWell plates, seeding iPSCs, and generating embryoid bodies (EBs) for subsequent differentiation studies.
Materials
AggreWell Plate (24-well format) - 1 plate
Matrigel (7-10 mg/mL concentration) - 2 mL
Falcon Tube (50 mL capacity) - 1 tube
Low-Attachment Plate (6-well format) - 1 plate
Pipette Tips (Sterile, 1000 µL) - As needed
Reagents:
Cell Culture Media Basal Media - Basic medium for cell culture
Cell Culture Media Complete Media - Basal media supplemented with growth factors
Enzyme Accutase - Enzyme for cell detachment
Buffer PBS - Phosphate-buffered saline for washing
ROCK Inhibitor Y-27632 - Inhibitor to prevent cell apoptosis
Chemical Emricasan - Caspase inhibitor for cell survival
Troubleshooting
Problem
Cells do not detach
Solution
Ensure Accutase is fresh and properly stored. Increase incubation time if necessary.
Problem
EBs are not forming
Solution
Check cell viability and ensure proper media conditions. Adjust seeding density if needed.
Safety warnings
Use personal protective equipment (PPE) including gloves and lab coats. Handle all reagents and cells according to institutional biosafety guidelines.
Preparing AggreWell Plates
1. Add 500 µL of Anti-Adherence Rinsing solution to each well of the 24-well AggreWell plate. 2. Centrifuge the plate at 1300g for 5 minutes with balance. 3. Rinse each well with 2 mL of warm basal media. 4. Add 1 mL of complete media to each well. Incubate the plate at 37°C while preparing cells for plating.
Cell Detachment
Aspirate media from the iPSC culture and wash with PBS
Add 1 mL of Accutase and incubate at RT for approximately 5 minutes until cells start to detach.
Add 1 mL of fresh complete StemFlex (with Y-27632) to each well to neutralize Accutase.
Collect cells into a Falcon tube and centrifuge at 300g for 5 minutes.
Cell Resuspension and Plating
1. Resuspend the cell pellet in 1 mL of fresh media and count viable cells using Trypan Blue.
Plate cells dropwise at a density of 2.5 million cells per AggreWell to achieve the desired number of cells per microwell
Add complete media to ensure a total of 2 mL of fresh media per well.
Centrifuge the plate at 100g for 3 minutes.
Generating Embryoid Bodies
Prepare Matrigel-coated plates by adding 2 mL of StemFlex per well.
Using special tips, transfer 1 mL of media from the AggreWell to a 50 mL Falcon tube.
Resuspend the remaining EBs by pipetting up and down and transfer to the Falcon tube.
Allow EBs to settle, then aspirate spent media gently.
Transfer cells from one AggreWell to all wells in the Matrigel-coated 6-well plate, ensuring EBs are equally distributed (aim for 30-40 EBs per well).
Generation of Hematopoietic Progenitor Cells (HPC) with HPC Media from STEMdiff Hematopoietic Kit
Next day is DAY 1: Remove StemFlex Medium and add 2mL Medium A (HPC Media from STEMdiff Hematopoietic Kit + Supplement A 1:200) per well of 6-well plate
DAY 3: Add 1 mL of Medium A per well
DAY 4: Aspirate media and add Medium B (HPC Media from STEMdiff Hematopoietic Kit + Supplement B 1:200)
DAY 6 and DAY 8: Add 1mL of Medium B
DAY 10-12: Collect HPCs
Firmly tap plate for ~60 seconds to dislodge attached HPCs; many should be floating already
collect all media and transfer to a 15mL tube
Count the cells: plate 150-200k cells per well of a matrigel-coated 6w plate
Microglia DifferenitaitonUntitled section
Resuspend HPC into iMG media supplemented with cytokines and plate on Matrigel coated plates
| COMPONENT | [CONC] | for 250 mL | for 500 mL | |
| Phenol-free* DMEM/F12 1:1 | 250 mL | 500 mL | ||
| GlutaMAX [100x] | 1x | 2.5 mL | 5 mL | |
| NEAA [100x] | 1x | 2.5 mL | 5 mL | |
| FILTER 0.22 MICRON | ||||
| ITS-G [100x] | 2x | 5 mL | 10 mL | |
| B27 [50x] | 2x | 10 mL | 20 mL | |
| N2 [100x] | 0.5x | 1.25 mL | 2.5 mL | |
| MTG [stock 11.5M, then secondary/intermediate stock of 0.1M in water] | 400uM | 1mL | 2 mL | |
| Insulin [stock 10mg/mL in water] | 5ug/mL | 125 uL | 250 uL |
Base media
| FACTOR | STOCK | CONCENTRATION | |
| IL34 | 100 ug/mL | 100 ng/mL (1:1000) | |
| TGFb | 50ug/mL | 50 ng/mL (1:1000) | |
| MCSF | 25 ug/mL | 25 ng/mL (1:1000) | |
| GM-CSF | 100ug/mL | 10 ng/mL (1:10000) | |
| CX3CL1* | 100ng/mL | 100 ng/mL | |
| CD200* | 100ng/mL | 100 ng/mL |
Cytokines to add fresh
*to add for final maturation after DAY23
EVERY OTHER DAY add 1ml iMG media with cytokines
When Cells are confluent or start to clump in the center of the well(depends on cell line) Salt into materigel coated plates
Collect some media from 6-well plate with 10mL pipette and transfer to 15/50mL tube, then add 1 mL DPBS and pipette up and down with a P1000 to dislodge stuck microglia from the plate (do it twice)
Spin collected media at 300 rcf for 5/7 min
Resuspend collected cell pellet in volume of SFM + MIT media
Count the cells and replate in a fresh matrigel plate at at least 1 million cells/plate
The SFM media will consist of 50% fresh iMG media and 50% of conditioned media (to save media and cytokine use)
Rock gently back and forth, put in incubator
Protocol references