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BRAIN Publication-derived

Intracellular energy controls dynamics of stress-induced ribonucleoprotein granules (eSG formation and SG clearance)

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

👤 Tao Wang, Xibin Tian, Han Byeol Kim, Yura Jang, Zhiyuan Huang, Chan Hyun Na, Jiou Wang ⏱ 130 days 📋 8 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Wang et al., 2022. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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