實驗概要
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.
主要試劑
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
主要設備
Desiccator
Iridectomy scissors
70-μm mesh cell strainer
Countess? Automated Cell Counter
實驗材料
Newborn rats or mice at 1?2 days after birth.
實驗步驟
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.