Materials and Reagents

Tissue culture dishes (60 x 15 mm) (Sigma-Aldrich, catalog number: P5237 )
Tissue culture dishes (100 x 20 mm) (Nunc, catalog number: 172958 )
Screw cap centrifuge tube (50 ml) (Sigma-Aldrich, catalog number: BR114821 )
24-well plates (Sigma-Aldrich, catalog number: CLS 3527 )
Pipette tips (10 μl, 200 μl and 1,000 μl) (Shineteh instruments co ltd., catalog number: PT4-W10 , PT1-Y200 and PT5-W10 )
Neonatal Sprague-Dawley pups (1-day-old)
Trypan blue (Thermo Fisher Scientific, Gibco^TM, catalog number: 15250061 )
Hanks’ Balanced Salt solution (HBSS) (Sigma-Aldrich, catalog number: 55021C )
Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761 )
Pyruvate (Sigma-Aldrich, catalog number: P2256 )
HEPES (Sigma-Aldrich, catalog number: H3375 )
Bovine Serum Albumins (BSA) (Sigma-Aldrich, catalog number: A9418 )
DMED powder (Thermo Fisher Scientific, Gibco^TM, catalog number: 12100-046 )
DMED/F-12, HEPES, no phenol red (Thermo Fisher Scientific, Gibco^TM, catalog number: 11039-021 )
Penicilline/Stretomycin (100x) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140-122 )
100 mM Sodium pyruvate solution (100x) (Thermo Fisher Scientific, Gibco^TM, catalog number: 11360-070 )
MEM non-essential amino acids solution (100x) (Thermo Fisher Scientific, Gibco^TM, catalog number: 11140-050 )
Fetal bovine serum (FBS) (NQBB, catalog number: A6806-11 )
Hank’s solution (see Recipes)
Dulbecco's modified Eagle’s medium (DMEM) (see Recipes)
Serum-free medium (100 ml) (see Recipes)

Equipment

37 °C, 5% CO₂ incubator (Water Jacketed Laboratory CO₂ Incubator)
Stereo microscope (Shineteh instruments co., catalog number: IH1-ZM150A )
Biological Inverted microscope (OLYMPUS CORPORATION, model: IX71 )
Mini micro centrifuge (Shineteh instruments co., catalog number: IC-MINIMAX )
Centrifuge (Hermle, catalog number: Hermle Z232K )
Water bath (Bioman Scientific Co Ltd, catalog number: SWB-20-1 )
Counting chamber (Shineteh instruments co., catalog number: PT14-901001 )
Operating scissors STR (Shineteh instruments co., catalog number: ST-014 )
Iris scissors STR (Shineteh instruments co., catalog number: ST-S009 )
Dressing forceps (Shineteh instruments co., catalog number: ST-D114 )
Iris forceps (Shineteh instruments co., catalog number: ST-I510 )
Tweezers forceps (Shineteh instruments co ltd., catalog number: ST-NO5 )
Pipetman (Gilson, catalog number: P10 , P200 and P1000)
Pipet-aid (Thomas Scientific, Flacon^TM, catalog number: 0410C04 )
Pipets (10 ml) (Tseng Hsiang Life Science ltd., catalog number: SP-1-C )
Ice bucket (Shineteh instruments co ltd., catalog number: PA8-4 )

Procedure

Primary neuron-glia cultures are prepared from cortical tissues of 1-day-old neonatal Sprague-Dawley pups.
After the rats are sacrificed, their brains are quickly removed aseptically. Under the stereo surgical microscope, the blood vessels and meninges are removed by tweezers.
Cerebral cortices (6~8 pups cerebral cortices/tube) are dissected under sterile conditions and kept on ice in conical tube containing 10 ml of Hank’s solution (without Ca²⁺ or Mg²⁺).
Remove Hank’s solution and add 15 ml of warm (37 °C) Dulbecco's modified Eagle’s media (DMEM) containing 10% heat-inactivated FBS to 50 ml conical tube.
Cortical cells are dissociated by trituration using a 10 ml pipette (about 15 times). Cells are centrifuged at 4 °C (1,500 x g) for 5 min to pellet cells.
After centrifugation, cells are re-suspended in 10 ml of DMEM containing 10% heat-inactivated FBS.
Dilute cells for counting by adding 20 μl of cells to 180 μl of culture medium (10x dilution).
Add 10 μl of the above cell dilution to a hemocytometer. Cells are counted in the 1 mm center square.
Cell density is determined by the average of the 5 randomly selected squares. To calculate the total # of cells/ml = (the average count per square) x (the dilution factor) x (10⁵/ml).
Dilute the 10 ml stock to make a final concentration of 5 x 10⁵ cells/ml.
To each well of 24-well culture plates 5 x 10⁵ cells are seeded in 0.5 ml of culture medium.
The cultures are incubated at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air.
Culture medium is replenished five days after plating, and is changed every three days thereafter.
The neuron-glial cells become confluent 10-12 days after plating.
Fourteen days in vitro (DIV14), the cultures are used for experiment. The cell composition is determined by immunostaining, followed by cell counting. The neuron-glia cultures consisted of approximately 37% ± 0.8% neurons, 51% ± 1.5% astrocytes, and 7% ± 7.7% microglia (Figure 1). In addition, cultures also consist of a small percentage (<5%) of other cells including oligodendrocytes, fibroblast and endothelial cells (data not shown).
The characterization of neuronal phenotypes is shown in Figure 2.

Figure 1. The composition of cell types in primary cortical neuron-glia cultures. Phase contrast photomicrograph of the primary cortical neuron-glia cultures in vitro (DIV14) (a), and representative immunocytochemically-stained photomicrographs using antibodies against (neuronal) dendritic marker microtubule associated protein 2 (MAP2) (b), astrocytic marker glial fibrillary acidic protein (GFAP) (c) or pan-macrophage lysosomal antigen (ED1) as a phagocytic marker for activated microglia (d). Percentage of immunostain positive cells of neurons (MAP2⁺), astrocytes (GFAP⁺) and activated microglia (ED1⁺) cells in cultures (e). Scale bar = 25 μm

Figure 2. Characterization of neuronal phenotype by immunocytochemical staining. Cultures were stained using antibodies against (a) neuronal marker neuronal nuclei antigen (NeuN) on 14 days in vitro (DIV 14), (b) excitatory amino acid carrier 1 (EAAC1) for glutamatergic neurons, (c) 5-hydroxytryptamine (5HT) for serotonergic neurons, (d) glutamate decarboxylase 65 (GAD 65) for GABAergic neurons, (e) choline acetyltransferase (ChAT) for cholinergic neurons, and (f) tyrosine hydroxylase (TH) for catecholaminergic neurons. Scale bar = 25 μm

Recipes

Hank’s solution (1,000 ml)
Reagents
Volume
Hanks’ balanced salt solution
1,000 ml
Sodium bicarbonate
0.35 g
Pyruvate
0.11 g
HEPES
4.76 g
Bovine serum albumins (BSA)
3 g
10% fetal bovine serum (FBS) / Dulbecco's modified Eagle’s medium (DMEM) (1,000 ml)
Reagents
Volume
DMED powder
1 package
Sodium bicarbonate
2.2 g
Pyruvate
0.11 g
HEPES
4.76 g
FBS
110 ml
Serum-free medium (100 ml) (drug treatment only)
Reagents
Volume
DMED/F-12, HEPES, no phenol red
97 ml
100x Penicilline/Stretomycin
1 ml
100x sodium pyruvate solution (100 mM)
1 ml
100x MEM non-essential amino acids solution
1 ml

Acknowledgments

This protocol is adapted from the previously published studies (Huang et al., 2015; Huang et al., 2014; Huang et al., 2009; Kim et al., 2000). These studies were supported in part by grants from the Hsin Sheng Junior College of Medical Care and Management (HSC-103-001 and HSC-104-015) and from Taipei Medical University (03C0720004A) to YNH; and from the Ministry of Science and Technology (MOST-104-2923-B-038-001-MY3 and MOST-104-2320-B-038-057-MY3) to JYW, Taiwan.

References

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