Cell Technology Topic: Rat Islet Cell Culture Experiment

Principle of experimental method
Peritoneal anesthesia with chloral hydrate was performed, and rat islets were isolated and purified by collagenase v retrograde perfusion, in situ digestion and Ficoll-400 gradient centrifugation, and islet cells were isolated and cultured.

Experimental material One week old Wistar rat
Reagents , kits D-Hanks' liquid trypsin type V collagenase glucose iodoacetic acid
Instruments , consumables, surgical scissors, ophthalmology scissors, ophthalmology, forceps, needles, surgical blades, petri dishes
Experimental steps <br> First, the experimental steps

1. One week old Wistar rats were sacrificed by cervical dislocation and soaked in 75% ethanol for 15 minutes. The pancreas was aseptically removed, rinsed in ice-cold sterile D-Hanks' solution, and the pancreas, capsule, blood vessels and other pancreas were carefully removed with ophthalmic scissors. The outer tissue was transferred to a penicillin vial.

2. Add a small amount of sterile D-Hanks' solution, cut into 0.1-1.0 mm3 pieces with ophthalmology scissors, gently aspirate the upper layer of small fat pieces and oil droplets with a dropper, and repeat with sterile D-Hanks' solution. Wash 8~10 times, add 10 times volume of sterile digestive enzyme solution [trypsin-collagenase digest: 0.05 g trypsin, 0.025 g type V collagenase (663 U/mg), 0.05 g glucose, dissolve in 100 mL A solution of Ca2+ and Mg2+ in 0.01 mol/L PBS (pH 7.4) was filtered through a 0.22 μm microporous membrane filter, digested at 38 °C ± 1 °C, and continuously turbulent during the digestion. After 10 minutes, the supernatant was discarded.

3. Wash the tissue block 2 to 3 times with sterile D-Hanks' solution, add fresh enzyme solution to continue digestion, repeat the above steps, and the edge of the tissue block is blurred.

4. Dip the tissue block into the digestive enzyme solution, digest it at 38 °C ± 1 °C for 10 minutes, separate the digestive enzyme solution from the tissue block, and re-add the fresh digestive enzyme solution for digestion. The original digestive enzyme solution is centrifuged at 1500 rpm for 10 minutes. Take the precipitate as the cells under digestion, resuspend with sterile D-Hanks', centrifuge, repeat 1~2 times, then wash the medium 2~3 times, and suspend the medium to obtain the cell suspension.

5. Repeat the digestion of the digested tissue block 5-6 times until the tissue block is completely digested. Repeat the above procedure.

6. Combine the obtained cell suspension several times, count, adjust the cell concentration to 2×105 cells/mL, inoculate the cell suspension in a 24-well plastic culture plate, 1 mL per well, and place at 37 ° C, 5% CO 2 , culture in a saturated humidity incubator.

7. Since fibroblast adherence is faster than islet cells, after 15 h of inoculation, gently shake the plate and connect the unattached cells to a new plate to remove some fibroblasts.

8. After incubating the cells in the new culture plate for 48 hours, the culture medium containing 2.5 ng/mL of iodoacetic acid is cultured for 5 h to remove most of the fibroblasts, while the islet cells are not damaged. The medium containing iodoacetic acid was washed twice, and the medium containing no iodoacetic acid was cultured in a 37 ° C, 5% CO 2 , saturated humidity incubator, and the culture medium was changed every 3 days to obtain a single layer of islet cells.

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