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中洪博元,细胞实验
z-bo
2020-07-20
702
As a biotechnology company with leading technology in cell culture experiments for many years, Zhonghong Boyuan has rich experience in both theoretical research and experimental operation. The following is a sharing of experimental methods and procedures for rat pancreatic islet cell culture. I hope it can help. Friends with experimental needs.
Principle of experimental method
Intraperitoneal anesthesia with chloral hydrate, using collagenase ⅴ retrograde perfusion, in situ digestion and Ficoll-400 gradient centrifugation methods to separate and purify rat pancreatic islets, and separate and culture islet single cells.
Experimental Materials
one week old Wistar rat
Reagents, kits D-Hanks’ solution Trypsin Type V collagenase Glucose Iodoacetic acid
Instruments, Consumables Surgical Scissors Surgical Forceps Ophthalmic Scissors Ophthalmic Tweezers Pins Surgical Blades Petri Dish
One, experimental procedure
1. One-week-old Wistar rats were sacrificed by breaking their necks, soaking in 75% ethanol for 15 minutes, removing the pancreas aseptically, rinsing in ice-cold sterile D-Hanks' solution, and carefully removing fat, capsules, blood vessels and other pancreas with ophthalmic scissors Transfer the outer tissue into a penicillin vial.
2. Add a small amount of sterile D-Hanks' solution, cut into 0.1-1.0 mm3 pieces with ophthalmic scissors, gently suck out the upper fine fat pieces and oil droplets with a dropper, and then use sterile D-Hanks' solution repeatedly Wash 8~10 times, add 10 times volume of sterile digestive enzyme solution [Trypsin-collagenase digestion solution: 0.05 g trypsin, 0.025 g type V collagenase (663 U/mg), 0.05 g glucose, dissolved in 100 mL without Ca2+, Mg2+ 0.01 mol/L PBS (pH 7.4) solution, use 0.22 µm microporous membrane to filter bacteria], digest at 38°C±1°C, shake continuously during the digestion process, discard the supernatant after 10 minutes.
3. Wash the tissue block with sterile D-Hanks’ solution 2~3 times, add fresh enzyme solution to continue digestion, repeat the above steps, at this time the edge of the tissue block is blurred.
4. Immerse the tissue block in the digestive enzyme solution, digest at 38℃±1℃ for 10 minutes, separate the digestive enzyme solution from the tissue block, add fresh digestive enzyme solution to the tissue block for digestion; and centrifuge the original digestive enzyme solution at 1500 rpm for 10 minutes , Take the precipitate to be the digested cells, resuspend in sterile D-Hanks', centrifuge, repeat 1~2 times, then wash 2~3 times with culture medium, and suspend in culture medium to obtain cell suspension.
5. Repeat the digestion of the digested tissue block 5-6 times until the tissue block is completely digested, repeat the above operation.
6. Combine the cell suspension obtained 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% CO2 , Cultivate in a saturated humidity incubator.
7. Since fibroblasts adhere more quickly than pancreatic islet cells, after 15 hours of inoculation, gently shake the culture plate and connect the non-adherent cells to the new culture plate to remove part of the fibroblasts.
8. After culturing the cells in the new culture plate for 48 hours, change to a freshly prepared medium containing 2.5 ng/mL iodoacetic acid and culture for 5 hours, which can remove most of the fibroblasts, while the islet cells are not damaged and cannot be replaced. Wash the medium containing iodoacetic acid twice, and change the medium without iodoacetic acid to culture in a 37°C, 5% CO2, saturated humidity incubator. Change the medium every 3 days to obtain a monolayer of islet cells.
Principle of experimental method
Intraperitoneal anesthesia with chloral hydrate, using collagenase ⅴ retrograde perfusion, in situ digestion and Ficoll-400 gradient centrifugation methods to separate and purify rat pancreatic islets, and separate and culture islet single cells.
Experimental Materials
one week old Wistar rat
Reagents, kits D-Hanks’ solution Trypsin Type V collagenase Glucose Iodoacetic acid
Instruments, Consumables Surgical Scissors Surgical Forceps Ophthalmic Scissors Ophthalmic Tweezers Pins Surgical Blades Petri Dish
One, experimental procedure
1. One-week-old Wistar rats were sacrificed by breaking their necks, soaking in 75% ethanol for 15 minutes, removing the pancreas aseptically, rinsing in ice-cold sterile D-Hanks' solution, and carefully removing fat, capsules, blood vessels and other pancreas with ophthalmic scissors Transfer the outer tissue into a penicillin vial.
2. Add a small amount of sterile D-Hanks' solution, cut into 0.1-1.0 mm3 pieces with ophthalmic scissors, gently suck out the upper fine fat pieces and oil droplets with a dropper, and then use sterile D-Hanks' solution repeatedly Wash 8~10 times, add 10 times volume of sterile digestive enzyme solution [Trypsin-collagenase digestion solution: 0.05 g trypsin, 0.025 g type V collagenase (663 U/mg), 0.05 g glucose, dissolved in 100 mL without Ca2+, Mg2+ 0.01 mol/L PBS (pH 7.4) solution, use 0.22 µm microporous membrane to filter bacteria], digest at 38°C±1°C, shake continuously during the digestion process, discard the supernatant after 10 minutes.
3. Wash the tissue block with sterile D-Hanks’ solution 2~3 times, add fresh enzyme solution to continue digestion, repeat the above steps, at this time the edge of the tissue block is blurred.
4. Immerse the tissue block in the digestive enzyme solution, digest at 38℃±1℃ for 10 minutes, separate the digestive enzyme solution from the tissue block, add fresh digestive enzyme solution to the tissue block for digestion; and centrifuge the original digestive enzyme solution at 1500 rpm for 10 minutes , Take the precipitate to be the digested cells, resuspend in sterile D-Hanks', centrifuge, repeat 1~2 times, then wash 2~3 times with culture medium, and suspend in culture medium to obtain cell suspension.
5. Repeat the digestion of the digested tissue block 5-6 times until the tissue block is completely digested, repeat the above operation.
6. Combine the cell suspension obtained 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% CO2 , Cultivate in a saturated humidity incubator.
7. Since fibroblasts adhere more quickly than pancreatic islet cells, after 15 hours of inoculation, gently shake the culture plate and connect the non-adherent cells to the new culture plate to remove part of the fibroblasts.
8. After culturing the cells in the new culture plate for 48 hours, change to a freshly prepared medium containing 2.5 ng/mL iodoacetic acid and culture for 5 hours, which can remove most of the fibroblasts, while the islet cells are not damaged and cannot be replaced. Wash the medium containing iodoacetic acid twice, and change the medium without iodoacetic acid to culture in a 37°C, 5% CO2, saturated humidity incubator. Change the medium every 3 days to obtain a monolayer of islet cells.