实验概要

Astrocytes are the  most numerous cell type in the central nervous system (CNS). They play  critical roles in adult CNS homeostasis, provide biochemical and  nutritional support of neurons and endothelial cells that form the  blood-brain barrier, perform the vast majority of synaptic glutamate  uptake, and maintain extracellular potassium levels. Astroglial  dysfunction has been implicated in a number of CNS pathologies. This  protocol describes the preparation of primary cortical astrocytes from  new-born rats or mice.

主要试剂

Ether

70% ethanol

Distilled water

Acetic acid

Collagen

0.05% Trypsin/EDTA solution

Hanks’ balanced salt solution (HBSS)

Gibco® Astrocyte Medium

EGF, Recombinant Human

Dulbecco’s Phosphate-Buffered Saline (D-PBS) without Ca² or Mg²

Dibutyryl cyclic-AMP (dBcAMP)

Penicillin-streptomycin

Trypan Blue Stain (included with the Countess® Automated Cell Counter) or LIVE/DEAD® Cell Vitality Assay Kit

主要设备

Desiccator

Iridectomy scissors

70-μm mesh cell strainer

Countess® Automated Cell Counter

实验材料

Newborn rats or mice at 1−2 days after birth.

实验步骤

1. Coat  the culture vessels with collagen and let stand for 45 minutes at room  temperature. Rinse with D-PBS without calcium or magnesium two times.

2. Anesthetize rat or mouse pups with ether in a desiccator in a chemical fume hood.

3. Remove  pups from the hood and spray 70% ethanol over the animal. Decapitate  the animals with scissors. Open the skull with iridectomy scissors.  Remove the meninges and dissect the brain tissue from the cortices.

4. Put  the cortices in a petri dish containing 5−10 mL of ice-cold HBSS. Pool  the cortices from two pups in a new petri dish and wash with 5 mL of  HBSS.

5.  Take the petri dish to a laminar flow hood. Mince the cortices into  small pieces with a scissors in a petri dish containing about 5 mL of  ice-cold HBSS. Transfer the tissue to a 15-mL sterile tube. Centrifuge  the tube at 200 × g for 3 minutes at 4°C and aspirate the supernatant.

6. Resuspend the tissue in 5 mL of 0.05% trypsin and incubate at 37°C for 25 minutes in a shaker bath.

7. Centrifuge  the tissue suspension at 200 × g for 3 minutes, aspirate the trypsin  solution with a pipette, and rinse the cells 3 times with 3 mL of HBSS.

8. Add 6 mL of astrocyte medium and pipet the cell suspension up and down with a 10 mL pipette to dissociate cells.

9. Filter  the cell suspension through a 70-μm mesh cell strainer into a 50-mL  sterile tube. Rinse the mesh with another 4 mL of astrocyte medium  (total of 10 mL suspension).

10. Remove 10 μL of the filtrate for counting on a hemacytometer or the Countess® Automated Cell Counter.

11. Determine the total number of cells and percent viability using trypan blue stain or the LIVE/DEAD® Cell Vitality Assay Kit.

12. Dilute the cell suspension to 5 × 10⁴ viable cells/mL with astrocyte medium and plate the cells into culture vessels at 2.5 × 10⁴/cm².

13. Incubate the cells in a 37°C incubator with 5% CO₂ and 90% humidity.

14. Change the astrocyte medium the next day and then every other day until cells are confluent.

15.  When confluent, feed the cells with astrocyte medium containing 0.25 mM  dBcAMP to induce differentiation. (Dilute 0.25 M stock of dBcAMP  1:1,000 in astrocyte medium.)

16. Feed the cultures with dBcAMP two times per week and check for differentiation.

17. Astrocytes are ready for experiments 2−3 weeks after culturing.