Neonatal Rat Primary Microglia: Isolation, Culturing, and Selected Applications

Mingwei Ni1, Michael Aschner1

1 Vanderbilt University Medical Center, Nashville, Tennessee
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 12.17
DOI:  10.1002/0471140856.tx1217s43
Online Posting Date:  February, 2010
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Microglial cells elaborate trophic factors and cytokines and remove toxins and debris from the extracellular space in the central nervous system, acting analogously to peripheral macrophages. Over the past two decades, increased attention has been directed at the role of microglia, not only in normal physiology, but also in mediating neurotoxicity. Activation of microglia is inherent to multiple neurodegenerative disorders and exposure to toxic compounds. In large measure, these revelations have come about as a result of technologies that enable researchers to obtain high yield and purity primary cultures of rodent microglia. The mechanical isolation protocol discussed in this unit offers an economical method to isolate large amounts of microglia in a short and not too labor‐intensive manner. Most importantly, it ensures a high yield of cells with great reproducibility. Given the ever‐increasing importance of microglia to the field of neurotoxicology research, the ability to isolate large quantities of primary microglia makes it possible to investigate the role and mechanisms associated with microglial modulation of neurotoxicity. We provide a detailed description on the methods that are routinely used in our laboratory for the isolation and culture of microglia, with emphasis on the steps that are deemed most critical for obtaining pure and healthy cultures. Curr. Protoc. Toxicol. 43:12.17.1‐12.17.16. © 2010 by John Wiley & Sons, Inc.

Keywords: microglia; isolation; culture; in vitro

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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation and Maintenance of Primary Microglial Cell Cultures
  • Support Protocol 1: Preparing Fire‐Polished and Sigmacote‐Treated Pipets for Cell Isolation
  • Support Protocol 2: Coating Cell Culture Plate with Poly‐L‐Lysine
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Isolation and Maintenance of Primary Microglial Cell Cultures

  • Newborn rat pups, PND 1
  • 70% (v/v) ethanol
  • Hank's buffered salt solution (HBSS; Sigma, cat. no. H‐2387)
  • Penicillin/streptomycin (Invitrogen, cat. no.15140‐163)
  • S‐MEM: Minimal Essential Medium with Earle's salts, modified for suspension cultures (Invitrogen, cat. no. 11385)
  • Dissociation medium (see recipe)
  • Growth medium (see recipe)
  • Deoxyribonuclease I (DNase I) from bovine pancreas, type IV (Sigma Chemical Company, cat. no. D‐5025); sterilize DNase I by passing it through a syringe filter with 25‐cm cellulose acetate membrane, 0.2‐µm pore size (Pall Corporation, cat. no. 4612)
  • 0.4% trypan blue staining solution, liquid, sterile‐filtered, cell culture tested (Sigma, cat. no, T8154)
  • Phosphate‐buffered saline (PBS; BD Biosciences, cat. no. 554781)
  • 1× trypsin
  • Autoclave
  • Surgical instrument tray, autoclavable (VWR, cat. no. 62687‐069)
  • 4 × 3–in. sterile dressing sponges (VWR, cat. no. 82004‐740)
  • Mayo scissors, 7‐in. length, 50‐mm curved blade (VWR, cat. no. 95039‐258)
  • Biohazard bags
  • Micro‐dissecting scissors, 4‐in. length, 25‐mm angled blade, 3 each (VWR, cat. no. 89049‐674)
  • Curved forceps, 4‐in. length, full curve, 0.8‐mm tip width, 2 each (VWR, cat. no. 82927‐392)
  • 100‐mm petri dish
  • Dissecting microscope
  • Curved forceps, 4‐in. length, full curve, 0.4‐mm tip width, 2 each (VWR, cat. no. 82027‐406)
  • Dumont forceps (Pattern #5), 110‐mm length, tip 0.1 × 0.06–mm, 2 each (VWR, cat. no 25719‐066)
  • 50‐ml polypropylene tubes, sterile (VWR, cat. no. 82018‐052)
  • 50‐ml beaker and stir bar (length, 25 mm), covered with foil and autoclaved
  • Pipet sterilizing metal boxes for 5 ¾ ‐in.
  • 9‐in Pasteur pipets, cotton‐plugged
  • Portable power pipet filler/dispenser
  • 10‐ml Sigmacote‐treated glass pipet (see protocol 2), sterile
  • Low‐speed (60 rpm) stir plate
  • Laminar‐flow cell culture hood with ultraviolet (UV) light
  • 15‐ml polystyrene centrifuge tubes, sterile (VWR, cat. no. 21008‐216)
  • 9‐in. cotton‐plugged Sigmacote‐treated Pasteur pipet, sterile
  • Low‐speed centrifuge (<1000 × g) with swinging‐bucket rotor and 50‐ml conical tube adapters
  • Hemacytometer
  • 5‐in. Pasteur pipets
  • 225‐cm2 vented cell culture flasks (BD Falcon, cat. no. 353138), sterile
  • CO 2 incubator (37°C, 95% room air/5% CO 2, 95% humidity; NAPCO series 8000DH, Thermo Scientific)
  • 1‐ml, 5‐ml, 10‐ml, and 25‐ml serological pipets, cotton‐plugged disposable polystyrene, individually wrapped (Midwest Scientific), sterile
  • Inverted phase‐contrast microscope
  • PRECISION Durafuge 200R (or equivalent) centrifuge with a swinging‐bucket rotor
  • 6‐well cell culture plate

Support Protocol 1: Preparing Fire‐Polished and Sigmacote‐Treated Pipets for Cell Isolation

  • Sigmacote (Sigma, cat. no. SL‐2)
  • 9‐in. Pasteur pipets
  • Bunsen burner
  • Cotton
  • Beaker
  • Paper towels
  • Pipet sterilizing metal boxes
  • Autoclave
  • 10‐ml glass serological pipets (VWR, cat. no. 20171‐042)

Support Protocol 2: Coating Cell Culture Plate with Poly‐L‐Lysine

  • Poly‐L‐lysine hydrobromide (Sigma, cat. no. P‐1274)
  • Borate buffer (see recipe)
  • Distilled water, cell culture tested (Invitrogen, cat. no. 15230‐147), autoclaved
  • S‐MEM: Minimal Essential Medium with Earle's salts, modified for suspension cultures (Invitrogen, cat. no. 11385)
  • 6‐well plates
  • Laminar‐flow cell culture hood with ultraviolet (UV) light
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Literature Cited

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