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BRAIN Publication-derived

Ischaemic Stroke Cortical Spheroids

Full SOP with Academic or a plan

Source McLaughlin et al. · Brown University, Providence, RI · 10.1101/2021.10.16.464587

Authors: Rachel M. McLaughlin, Amanda Laguna, Ilayda Top, Christien Hernandez, Liane L. Livi, Liana Kramer, Samantha Zambuto, Diane Hoffman-Kim Duration: 16 days 11 phases Cell source: Primary Postnatal Rat Cortex (CD rats, postnatal day 0-2)

Abstract

This protocol describes the generation and culture of three-dimensional cortical spheroids from primary postnatal rat cortex, and their exposure to ischemic injury conditions (glucose deprivation, oxygen deprivation, or combined oxygen-glucose deprivation) to model stroke pathology. The spheroids reproduce key features of ischemic brain injury including ATP depletion, cytoskeletal disruption, astrogliosis, neurovascular dysfunction, and neural network disruption, and can be used for therapeutic screening.

Cell source
Primary Postnatal Rat Cortex (CD rats, postnatal day 0-2)
Application
Disease modeling

Protocol overview

66 steps across 11 phases

Phase 1 · Cell Isolation and Preparation Day 0
  1. a Harvest postnatal rat cortex
  2. b Dissect cortical tissue in Hibernate A buffer
  3. c Enzymatic digestion with papain
  4. d Remove papain and triturate tissue
  5. e Filter and centrifuge cell suspension
  6. f Resuspend in complete cortical medium
  7. g Additional filtering and centrifugation cycles
  8. h Determine cell viability and count
  9. i Prepare 2D viability control cultures
Phase 2 · 3D Spheroid Culture Setup Day -7 to Day 0
  1. a Prepare agarose gel molds
  2. b Remove agarose gels from molds
  3. c Equilibrate agarose gels with incomplete medium
  4. d Final equilibration with complete cortical media
Phase 3 · Spheroid Seeding and Early Culture Day 0
  1. a Prepare cell suspension for seeding
  2. b Seed cells into microwells
  3. c Allow cells to settle into microwells
  4. d Add complete cortical medium
  5. e Change medium at 48 hours
  6. f Maintain ongoing medium changes
Phase 4 · Ischemic Injury Induction Day 13-14
  1. a Prepare spheroids for deprivation
  2. b Control condition setup
  3. c Glucose deprivation (GD) condition setup
  4. d Oxygen deprivation (OD) condition setup
  5. e Oxygen-glucose deprivation (OGD) condition setup
  6. f Maintain deprivation conditions for 24 hours
Phase 5 · ATP Assay Day 14-15 (post-deprivation)
  1. a Extract spheroids from agarose gels
  2. b Transfer spheroids to assay plate
  3. c Add CellTiter-Glo 3D Reagent
  4. d Induce cell lysis
  5. e Measure luminescence
Phase 6 · Immunohistochemistry and Imaging Day 14-15 (post-deprivation)
  1. a Fix spheroids
  2. b Wash fixed spheroids
  3. c Store fixed spheroids
  4. d Prepare 3D blocking solution
  5. e Permeabilize and block spheroids
  6. f Incubate with primary antibodies
  7. g Wash with PBT after primary incubation
  8. h Blocking solution wash
  9. i Incubate with secondary antibodies
  10. j Wash with PBT after secondary incubation
  11. k DAPI nuclear staining
  12. l Final PBS wash
  13. m Optical clearing with FOCM (optional, for laminin and GFAP imaging)
  14. n Prepare samples for imaging
Phase 7 · Confocal Imaging Day 15-16
  1. a Set up confocal microscope
  2. b Select imaging objective
  3. c Acquire fluorescent images
  4. d Capture z-stacks
  5. e Convert and process image stacks
Phase 8 · LIVE/DEAD Viability Assay Day 14-15 (post-deprivation)
  1. a Incubate spheroids with LIVE/DEAD reagents
  2. b Wash spheroids
  3. c Transfer to confocal imaging dish
  4. d Image at 37°C
Phase 9 · Calcium Imaging Day 8-9
  1. a Incubate spheroids with calcium indicator
  2. b Wash spheroids
  3. c Transfer to confocal imaging dish
  4. d Maintain temperature during imaging
  5. e Acquire time-lapse video
Phase 10 · Image Analysis Day 16+
  1. a GFAP quantification
  2. b Laminin/capillary-like network quantification
  3. c Calcium imaging analysis
Phase 11 · N-acetylcysteine (NAC) Treatment Day 13
  1. a Prepare NAC solutions
  2. b Add NAC and induce control condition
  3. c Add NAC and induce OGD condition
  4. d Maintain treatment for 24 hours
  5. e Perform ATP assay

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Attribution

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

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