Culture of cerebellar granule cells
Culture of cerebellar granule cells
Cerebellums from 4-8 neonatal rats were cut into small cubes, placed in HBSS, and digested with trypsin for 15 min at 37℃. Cells were inoculated into petri dishes or culture flasks coated with L-polylysine.
Operation method
Program 23.39 Cultures of cerebellar granule cells
Principle
Cerebellums from 4-8 neonatal rats were cut into small cubes, placed in HBSS, and digested with trypsin for 15 min at 37°C. Cells were inoculated into petri dishes or culture flasks coated with L-polylysine.
Materials and Instruments
DMEM HBSS L-polylysine Cytarabine 0.025% Trypsin Silicone Move 1. The cerebellum was excised under sterile conditions and placed in HBSS (containing 3 g/L BSA). For more product details, please visit Aladdin Scientific website.
P e tr i Petri dishes Dissecting knives Dissecting scissors Dissecting forceps Pasteur pipettes 10 ml pipettes Test tubes Water baths
2. The brain tissue was cut into cubes of approximately 0.5 mm3 size using a scalpel.
3. Transfer the tissue block into a 12 ml test tube and wash three times with HBSS. After each wash, let the block sink to the bottom of the tube.
4. Add 10 ml of 0.025 % trypsin (prepared in HBSS) and incubate for 15 min at 37°C in a water bath.
5. Transfer the trypsin-digested brain tissue into a 50 ml centrifuge tube and add 20 ml of growth medium to terminate trypsin digestion.
6. Disperse the tissue by grinding with a siliconized Pasteur pipette until a single cell suspension is obtained.
Siliconized Pasteur pipette:
(a) Dilute the silicone solution to 0.1% to 1% with ultrapure water;
(b) Immerse the straw in silicone liquid or rinse the inner surface of the straw with silicone liquid;
(c) Leave the pipette to dry for 24 h or for a few minutes at 100°C to dry;
(d) Sterilize the straws by dry heat.
Treat Petri dishes with L-polylysine:
(a) Dissolve L-polylysine (10 mg/L) in ultrapure water;
(b) Filter the mixture to remove bacteria;
(c) 1 ml of L-polylysine solution was added to each 35 mm Petri dish;
(d) The L-polylysine solution was aspirated after 10-15 min and 1-15 ml of culture medium was added;
(e) Leave the Petri dishes in the incubator for more than 2 h until the cells were inoculated.
7. Place the centrifuge tube containing the cell suspension for 3~5 min to allow small tissue clumps to sink to the bottom of the tube. Then, use a Pasteur pipette to aspirate the tissue mass.
8. Centrifuge the single cell suspension (200 g for 5 min) and aspirate the supernatant.
9. Suspend the cell mass with growth medium and then inoculate the cells at a density of 2. 5×106~3. 0×106 cells per dish.
10. After incubation for 2~4 d (best after two days), add 5~10 μmol/L cytarabine, and continue incubation for 24 h.
11. Change the solution with DMEM (containing 30 mmol/L glucose, 2 mmol/L glutamine, 24.5 mmol/L potassium chloride, 100 mU/L insulin (Sigma, Ⅰ-1882), 7 μmol/L p-aminobenzoic acid (Sigma, A-3659), 100 μg/ml gentamicin, and 10 % heat-inactivated FCS).