Cell Technology Topic: Rat Star Glial Cell Culture Experiment

First, the experimental steps

V. Results

Figure 3: Cell growth curve

1. Take one of 14.5d SD rats.

Second, phenobarbital 0.5 ml intraperitoneal injection, placed on the operating table after anesthesia, alcohol cotton ball disinfection of the skin, open the uterus (about 12-14), into the anatomy, cut open the uterus, remove the embryo , placed in the anatomical solution, disconnected between the ear and eye, take the side of the head tissue, remove the meningeal tissue, take the original midbrain tissue, into the anatomical solution of the centrifuge tube, after all taken out, 1500 rpm 2 minute.

3. Aspirate the supernatant with a pipette and add a trypsin tube (1 ml/vial) to the tissue cell pellet at room temperature for 30 minutes, 1500 rpm, and centrifuge for 5 minutes (the anatomical solution can be added to terminate the trypsin before centrifugation).

4. Aspirate the supernatant, add a tube of Dnase (2 ml/vial) to the tissue cell pellet, react at 37 ° C for 10 minutes, centrifuge at 1500 rpm, centrifuge for 5 minutes, aspirate the supernatant, and add 2 ml of DM to the tissue cell pellet. The culture medium is blown, counted (counting formula is (N1+N2+N3+N4)/4*10 ^₄ ), cultured in a culture flask at 3*10 ⁵ / bottle, and marked (time, original or the first few Substitute generation, name, cell name, etc.), and cultured in an incubator.

I. Isolation of the neocortex of rat embryos (16-18 weeks), placed in L-15 (or Hank's balanced salt / DMEM solution, no Hepes) medium, remove meninges and blood vessels, hippocampus, caudate nucleus and other non-cortical tissues .

2. Cut it into 1 mm ³ tissue blocks with a scalpel.

3. Transfer 250 μl of collagenase stock solution (1.33%, dissolved in L-15) to 1 ml of L-15 (or D-MEM) using a 1 ml tip. The collagenase solution was diluted with 1-1.5 ml for every 2 brain tissue blocks.

4. Incubate for 30 min in a 37 ° C water bath.

5. Centrifuge for 5 min at 1000 rpm (approximately 230 g) or centrifuge for 1 min at 3 000 rpm (approximately 2 000 g).

6. Go to the supernatant.

7. Resuspend the cells in a solution containing EDTA-CMF and mix with trypsin at a ratio of 1:4 to 1:10. The ratio of the two can be appropriately adjusted depending on the observed cell digestion.

8. Add trypsin inhibitor and DNase solution and mix for 5 minutes.

Centrifuge for 5 min at 9000 rpm (approximately 230 g) or centrifuge for 1 min at 3 000 rpm (approximately 2 000 g).

Ten, go to the supernatant. Add 500 μl D-MEM-BS.

Eleven, gently blow into a single cell suspension, start to blow 10 times with a 5 ml gun, and use a 18-gauge needle to blow when needed. (Note: Do not create air bubbles.) 200 mesh / 300 mesh nylon mesh filter.

12. Transfer the cells to a culture flask containing 10% FCS-DMEM, culture a culture flask with a density of 2× ^{10 6} /25 cm ^{2 , and a} culture flask of 4-6 x 10 ⁶ /75 cm ³ . Incubate at 37.2 ° C, 37.5%.

Thirteen, change the liquid on the second day, and then change the liquid every three days.

Fourteen, 7-10 days, the cells fuse.

15. After the cells are fused, the caps are sealed with a sealing film and cultured on a trajectory shaker. The rotation speed is preferably such that no bubbles are generated. Shake overnight at 37 °C. The cells are protected from light.

16. Remove the supernatant and add fresh 10% FCS-DMEM.

17. Add 200 μl of 1 mM cytarabine/10 ml medium. Incubation was carried out for 48 hours at 37.2 ° C, 37.5% to eliminate dividing cells.

18. Replace with fresh 10% FCS-DMEM and incubate for 24 hours.

Nineteen, repeat steps 17-18 to eliminate all dividing cells