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Protocol for Culturing Rat Microglia

Table of Contents

Introduction

Welcome to the lab! If you're looking to investigate the underlying mechanisms of neuroinflammation or neurodegenerative diseases, cultivating primary microglia is a fantastic starting point. As the resident immune sentinels of the central nervous system, microglia are essential for maintaining tissue homeostasis, clearing out debris, and remodeling synaptic connections. When triggered by disease or injury, these cells dramatically change their morphology, ramp up their phagocytic activity, and release potent proinflammatory signals. This straightforward, easy-to-follow guide will walk you through the process of isolating and culturing rat microglia from postnatal day 1 or 2 (P1-P2) cortical tissue. Let's dive in and get those cultures growing!

Materials

  • 70% ethanol
  • Poly-L-Lysine
  • Sterile deionized distilled H2O (ddH2O)
  • DNase I
  • Fetal Bovine Serum (FBS)
  • L-glutamine-penicillin-streptomycin solution (100x)
  • HBSS, with Ca2+ and Mg2+, phenol red-free (1x)
  • Minimum Essential Medium Eagle (MEM)
  • !!! **PBS (HB8088)** !!!
  • Trypan blue (0.4%)
  • Trypsin solution from porcine pancreas (10x)
  • P1-P2 rat pups
  • Ice
  • 5, 10, 25, and 50 mL pipettes & pipette controller
  • 50 mL conical centrifuge tubes
  • T175 sterile cell culture flasks
  • 40 µm cell strainers
  • 100 x 20 mm petri dishes
  • Pipette tips
  • 0.22 µm syringe filters
  • 37°C, 5% CO2 humidified incubator
  • 37°C water bath
  • Centrifuge
  • Dissecting and inverted microscopes
  • Hemocytometer
  • Dissection tools (fine straight forceps, curved forceps, micro spatula, large surgical scissors)
  • Orbital shaker

Media & Buffers

Component Concentration
Co-Culture Media  
Minimum Essential Medium Eagle (MEM) Base
Fetal Bovine Serum (FBS) 10%
L-glutamine-penicillin-streptomycin solution 1x
Component Concentration
DNase I Stock Solution  
DNase I 3 mg/mL
!!! **PBS (HB8088)** !!! Solvent

Sterilize the DNase I Stock Solution by passing it through a 0.22 µm syringe filter. This prepared solution may be stored at ≤ -20°C for up to 2 years.

Protocol

Part 1: Preparation of Cell Culture Flasks

Note: Perform all flask preparation steps under strict aseptic conditions within a laminar flow cell culture hood.

  1. Prepare a 100 µg/mL working solution of poly-L-lysine by diluting the stock reagent with !!! **PBS (HB8088)** !!!.
  2. Dispense 10 mL of the 100 µg/mL poly-L-lysine solution into each T175 cell culture flask. Gently rock the flasks side-to-side to ensure even and complete coverage of the growth surface.
  3. Incubate the coated flasks in a humidified environment at 37°C and 5% CO2 for a minimum of 20 minutes.
  4. Aspirate the poly-L-lysine solution. Rinse the interior surfaces of the flasks twice using sterile ddH2O. Fully aspirate any remaining liquid and allow the flasks to dry completely before use. Prepared flasks may be stored at 2-8°C for up to 2 months.

Part 2: Dissection of Rat Cortical Tissue

Note: Submerge all dissection instruments in 70% ethanol for at least 10 minutes prior to use. Allow the tools to air dry completely over a sterile field.

  1. Pre-chill three 100 x 20 mm petri dishes and one 50 mL conical tube containing !!! **PBS (HB8088)** !!! on ice. Add 8 mL of HBSS to a separate 50 mL conical tube (designated as the Dissociation Tube) and keep it on ice.
  2. Euthanize P1-P2 rat pups by decapitation at the cervicomedullary junction using large surgical scissors.
  3. Submerge the severed heads in the chilled 50 mL conical tube containing PBS. Maintain the tube strictly on ice.
  4. Transfer an individual head to one of the chilled petri dishes containing PBS.
  5. Immobilize the head using curved and fine forceps. Utilizing small surgical scissors, execute shallow incisions through the cranium progressing from caudal to rostral, taking great care to avoid mechanical damage to the underlying neural tissue.
  6. Gently peel back the bisected halves of the skull to expose the brain.
  7. Extract the intact brain with a micro spatula and transfer it into the second chilled petri dish containing PBS. Repeat the extraction procedure for the remaining heads.
  8. Using forceps, cleanly bisect the hemispheres along the median longitudinal fissure. Excise and discard the olfactory bulbs, brainstem, and cerebellum. Relocate the isolated forebrains to the third chilled petri dish.
  9. Under a dissecting microscope, carefully strip away the meninges, midbrain, and hippocampus from each hemisphere. Deposit the isolated cortical tissue directly into the Dissociation Tube containing 8 mL of chilled HBSS.

Part 3: Cell Dissociation and Culturing

Note: All subsequent procedures involving tissue, media, or open vessels must be executed within a laminar flow cell culture hood.

  1. Pre-warm the Co-Culture Media in a 37°C water bath. Allow the 10x Trypsin and DNase I Stock Solutions to equilibrate to room temperature.
  2. Introduce 400 µL of 10x Trypsin and 200 µL of DNase I Stock Solution into the Dissociation Tube containing the cortical tissue. Triturate the tissue gently using a 25 mL pipette to fragment it into segments approximately 1 mm2 in size. Avoid over-mincing, which generates excess debris and compromises culture viability.
  3. Gently agitate the Dissociation Tube for 5 seconds, followed by a 15-minute incubation in the 37°C water bath.
  4. Remove the tube from the water bath and allow it to rest undisturbed at room temperature for 5 minutes to permit tissue sedimentation.
  5. Carefully transfer the supernatant (leaving approximately 3 mL behind) into a fresh 50 mL conical tube designated as the Collection Tube. Immediately supplement the Collection Tube with 2 mL of FBS, mix by swirling gently, and maintain at room temperature. Use plastic or siliconized glass pipettes exclusively to prevent microglial adhesion.
  6. To the residual tissue in the Dissociation Tube, add 9 mL of HBSS, 400 µL of 10x Trypsin, and 200 µL of DNase I. Triturate the tissue with a 10 mL pipette to achieve further cellular dissociation.
  7. Swirl the tube gently for 5 seconds. Incubate at 37°C for 15 minutes, manually agitating the suspension every 5 minutes during the incubation period.
  8. Remove the tube from the water bath and allow sedimentation at room temperature for 5 minutes.
  9. Decant the resulting cell suspension into the Collection Tube, pooling it with the initial harvest. Do not transfer any settled debris pellets.
  10. Introduce 10 mL of !!! **PBS (HB8088)** !!! to the remaining tissue fragments in the Dissociation Tube. Swirl gently and allow the contents to settle for 5 minutes.
  11. Transfer the PBS wash into the Collection Tube. Centrifuge the pooled cell suspension at 300 x g for 5 minutes at room temperature. Decant the supernatant with extreme care, as the cell pellet remains highly fragile at this stage.
  12. Wash the cell pellet by gently resuspending in 25 mL of !!! **PBS (HB8088)** !!!. Centrifuge again at 300 x g for 5 minutes at room temperature and cleanly discard the supernatant.
  13. Reconstitute the final cell pellet in 5 mL of pre-warmed Co-Culture Media. Prepare two serial 1:5 dilutions of the cell suspension utilizing 0.4% Trypan blue, and determine the viable cell concentration utilizing a standard hemocytometer.
  14. Seed approximately 1.2 x 10^6 viable cells per pre-coated T175 flask, supplementing each with 45 mL of Co-Culture Media. Incubate the flasks at 37°C in a 5% CO2 humidified atmosphere. This initial seeding constitutes Day 0 of the culture schedule.

Part 4: Culture Maintenance and Microglia Harvest

Note: Microglia preferentially thrive in nutrient-depleted conditions compared to neurons. Do not perform any media changes or feed the culture following Day 7.

  1. Maintain the cultures according to the following schedule, performing complete media exchanges with pre-warmed Co-Culture Media on designated days:
    • Day 1: Cells begin to adhere, separate, and extend processes.
    • Day 2: Emergence of astrocytes exhibiting a large, flattened morphology.
    • Day 4: Astrocytes reach 2-6% confluency. Perform a complete media change.
    • Day 7: Astrocytes reach 60-80% confluency. Perform a final complete media change.
    • Days 9-10: Astrocytes attain 100% confluency.
    • Days 11-12: Target window to harvest microglia.
  2. To execute the harvest, secure the T175 flasks onto an orbital shaker housed within a 37°C, 5% CO2 incubator. Agitate the flasks at 180 rpm for exactly 2 hours to selectively detach the microglia.
  3. Collect the Co-Culture Media containing the suspended microglia and filter the fluid through a 40 µm cell strainer into a 50 mL conical tube. Repeat this process for all available flasks.
  4. Centrifuge the collected media at 300 x g for 5 minutes at room temperature. Decant and discard the supernatant.
  5. Resuspend the isolated microglial pellet in the desired assay media required for downstream applications. Typical yields approximate 2-6 million microglia per T175 flask.

References

Ni, M., & Aschner, M. (2010). Neonatal Rat Primary Microglia: Isolation, Culturing, and Selected Applications. Current Protocols in Toxicology, Chapter 12, Unit 12.17.1-16.