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BRAIN Publication-derived

Amyotrophic Lateral Sclerosis Brain Models for Disease Modelling

Full SOP with Academic or a plan

Source Wang et al., 2022 · Johns Hopkins University · 10.1038/s41467-022-33079-1

Authors: Tao Wang, Xibin Tian, Han Byeol Kim, Yura Jang, Zhiyuan Huang, Chan Hyun Na, Jiou Wang Duration: 130 days 8 phases Cell source: Human iPSC-derived cortical organoids and motor neurons; HeLa cells; Patient-derived iPSC (C9orf72-ALS)

Abstract

This protocol establishes methods to induce and characterize energy deficiency-induced stress granules (eSGs) and to monitor stress granule (SG) clearance under various metabolic stress conditions. The protocol enables identification of ATP-dependent regulation of RNP granule assembly and disassembly, with applications to understanding amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) pathogenesis.

Cell source
Human iPSC-derived cortical organoids and motor neurons; HeLa cells; Patient-derived iPSC (C9orf72-ALS)
Application
Disease modeling; Mechanistic study of stress granule dynamics in energy deficiency

Protocol overview

30 steps across 8 phases

Phase 1 · Cell preparation and glycolysis inhibition Day 0
  1. a Prepare cells for glycolysis inhibition
  2. b Induce glycolysis blockade via two methods
  3. c Incubate cells under glycolytic blockade
Phase 2 · OXPHOS inhibition and low-glucose challenge Day 0
  1. a Prepare cells for OXPHOS inhibition
  2. b Add oligomycin to block ATP synthase
  3. c Incubate under OXPHOS inhibition
Phase 3 · Conventional SG induction by arsenite Day 0
  1. a Treat cells with arsenite
  2. b Verify SG formation by live-cell imaging or immunofluorescence
Phase 4 · SG clearance assay Day 0
  1. a Induce conventional SGs with arsenite
  2. b Remove arsenite and initiate recovery in energy-deficiency media
  3. c Assess SG clearance by immunofluorescence
Phase 5 · ATP measurement and verification Day 0
  1. a Extract ATP from cells
  2. b Measure protein concentration
  3. c Quantify ATP using luciferase-based luminescence assay
Phase 6 · Immunofluorescence and image analysis Day 0
  1. a Fix cells
  2. b Permeabilize and block
  3. c Incubate with primary antibodies
  4. d Wash and apply secondary antibodies
  5. e Mount and image
Phase 7 · iPSC-derived cortical organoid and motor neuron generation (if applicable) Days 0-130
  1. a Prepare iPSCs for organoid induction
  2. b Generate uniform spheroids (Day 0)
  3. c Initiate neural induction (Days 0-6)
  4. d Neural differentiation phase (Days 6-25)
  5. e Neuronal maturation phase (Days 25-43+)
  6. f Perform stress treatments on organoids (Days 100-130)
Phase 8 · Organoid cryosectioning and immunofluorescence After treatment
  1. a Fix organoids
  2. b Cryoprotect organoids
  3. c Embed and freeze organoids
  4. d Cryosection organoids
  5. e Immunofluorescence staining of organoid sections

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Attribution

This SOP was written by Organthis based on the published method in Wang et al., 2022. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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