Isolation and Culture of Microglia from Rat Brains using Serum-Free Media

Abstract

Source: Collins, H. Y., et al., Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium. J. Vis. Exp. (2018)

This video demonstrates a method for isolating and culturing microglia from rat brains. It entails separating microglia from other neuronal and non-neuronal cells using plates coated with a monoclonal antibody specific to microglia.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Prepare a Petri Dish for CD11b Immunopanning (Day 0)

NOTE: Prepare 1 immunopanning dish for every 1 - 2 juvenile rat brains.

1. Add 25 mL of 50 mM Tris pH 9.5 solution to a 15-cm Petri dish.
2. Add goat anti-mouse IgG (H+L chains) to the dish for a final concentration of 6 µg/mL. Swirl plate to evenly distribute.
3. Incubate the dish for 1 - 3 h at 37 °C.
4. Rinse dishes three times with DPBS++ (phosphate-buffered saline (PBS....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Douncing Buffer			Recipe: 200 μL of 0.4% DNaseI in 50 mL of DPBS++ Comments: Use when ice-cold
Panning Buffer			Recipe: 2 mg/mL of peptone from milk solids in DPBS++
Microglia Growth Medium (MGM)			Recipe: DMEM/F12 containing 100 units/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine, 5 μg/mL N-acetyl cysteine, 5 μg/mL insulin, 100 μg/mL apo-transferrin, and 100 ng/mL sodium selenite Comments: Use ice-cold MGM to pan microglia off of immnopanning dish.
Collagen IV Coating			Recipe: MGM containing 2 μg/mL collagen IV
Myelin Seperation Buffer			Recipe: 9 mL Percoll PLUS, 1 mL 10x PBS without Ca++ and Mg++, 9 μL 1 M CaCl2, 5 μL 1 M MgCl2 Comments: Mix well after the addition of CaCl2 and MgCl2
Trypsin	Sigma	T9935
DMEM/F12	Gibco	21041-02
Penicillin/ Streptomycin	Gibco	15140-122
DMEM (high glucose)	Gibco	11960-044
Collagen IV			Reconstitution: 200 μg/mL in PBS Concentration used: 1:100 Storage: -80°C
mouse anti-rat CD11b monoclonal (clone OX42)	Bio-Rad	MCA275R	Panning: 1:1,000; Staining: 1:500
TGF-b2/IL-34/Cholesterol containing growth media (TIC)			Recipe: MGM containing human 2 ng/mL TGF-b2, 100 ng/mL murine IL-34, 1.5 μg/mL ovine wool cholesterol, 10 μg/mL heparan sulfate, 0.1 μg/ml oleic acid, and 0.001 μg/ml gondoic acid Comments: Make sure to add cholesterol to media warmed to 37 °C and do not add more than 1.5 μg/mL or it will precipitate out. Do not filter cholesterol-containing media. Equilibrate TIC media with 10% CO2 for 30 min- 1 hr before adding to cells to insure optimal pH.
Percoll PLUS	GE Healthcare	Cat# 17-5445-02
Primaria Plates	VWR	Cat# 62406-456
DNaseI	Worthington	Cat# DPRFS