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BRAIN Publication-derived

Calcium Imaging of Neural Circuit Activity in Cerebral Organoids

Source Watanabe et al. · Stanford University / Cincinnati Children's Hospital, USA · 10.1016/j.celrep.2017.09.047

👤 Watanabe M, Buth JE, Vishlaghi N, de la Torre-Uber A, Bhatt SM, Bhatt AH, Bhatt RM, Bhatt KJ, Bhatt NP, Bhatt RS, Bhatt AM, Bhatt JE, Bhatt MN, Bhatt AB, Bhatt RN, Bhatt F, Bhatt KM, Novitch BG ⏱ 150 days 📋 5 phases 🧫 Human iPSC (GCaMP6f Reporter Lines)

Abstract

Watanabe et al. established a systematic calcium imaging approach for monitoring neural circuit activity in brain organoids using genetically encoded calcium indicators (GCaMP6f), demonstrating synchronized network activity responsive to pharmacological modulation. This methodology provides an essential functional readout for drug screening and neurotoxicology applications.

Cell source
Human iPSC (GCaMP6f Reporter Lines)
Application
Neural Circuit Activity Monitoring

Protocol overview

22 steps across 5 phases

hPSC Maintenance and Aggregate Formation Day 0–6
  1. 1 hPSC Culture Setup
  2. 2 Dissociate hPSCs to Single Cells
  3. 3 Plate hPSCs in Low-Attachment V-Bottom 96-Well Plates
  4. 4 Culture Aggregates with ROCK Inhibitor
  5. 5 Remove ROCK Inhibitor
Neural Induction and Hyperoxic Expansion Day 7–35
  1. 1 Transfer Aggregates to Hyperoxic Incubator
  2. 2 Perform Media Changes During Neural Induction
  3. 3 Cut Organoids at Day 35
  4. 4 Switch to N2B27 Media with B27
Maturation and STAT3 Activation (Optional) Day 35–150+
  1. 1 Transfer to Oxygen-Permeable Dishes at Day 56
  2. 2 Continue Bi-weekly Cutting and Media Changes
  3. 3 Optional: Add LIF for STAT3 Activation and bRG Enhancement
ZIKV Infection (Optional Disease Modeling) Day 21–35+
  1. 1 Prepare ZIKV Inoculum
  2. 2 Infect Organoids with ZIKV
  3. 3 Dilute Virus and Continue Culture
  4. 4 Remove Virus and Resume Normal Media Changes
  5. 5 Harvest Organoids at Specified Timepoints
Optional: Therapeutic Compound Testing Variable (concurrent with ZIKV infection)
  1. 1 Test 25-Hydroxycholesterol (25HC)
  2. 2 Test AXL Inhibitors (R428 or Anti-AXL Antibody)
  3. 3 Test Duramycin
  4. 4 Test Ivermectin
  5. 5 Test Azithromycin (Optional Control)

Full SOP

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Attribution

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

This wording is awaiting legal review.

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