Mouse Pancreatic Organoid Culture Medium Kit
Mouse Pancreatic Organoid Culture Medium Kit
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Product Specification
| Usage |
1. Primary (1) The collected tissue must be placed in a sampling bottle of pre-cooled (2-8 °C) tissue preservation solution E, and quickly transported to a clean laboratory for tissue processing and cell separation, photographed and registered information. (2) Prepare several petri dishes, and add primary culture buffer B pre-cooled at 4 °C for later use. (3) Sterilize the sampling bottle, put the tissue in a petri dish, wash it three times with primary culture buffer B, and cut the tissue into a volume of about 1-3mm with ophthalmic scissors or scalpel3The tissue block. (4) The tissues were digested with mouse normal pancreatic primary tissue digestive juice C, and digested by shaking at 37 ℃ for 10-20min (the digestion situation was observed at any time during digestion). (5) Take a small amount of liquid and observe it under a microscope. After more single cells or cell clusters below 70um are observed under the microscope, add triploid volume primary culture buffer B to terminate digestion. (6) Filter with a sieve with a pore size of 100um, collect the filtrate, enrich and centrifuge at 300g for 5 minutes, remove the supernatant, add primary culture buffer B and re-suspend and centrifuge. (7) Matrigel calculation: After step 6, observe the collected tissue volume, add 25 times the tissue volume of Matrigel (abs9495) to resuspend and plate. (8) Take a 24-well cell culture plate as an example. Each well is dispensed with 25ul tissue matrigel mixture for plating (operation at 4 °C). (9) Put the laid culture plate into a 37 ℃ incubator for 10-15 minutes to gel, and add mouse normal pancreatic organoid medium A (restored to room temperature) for culture. 2. Organoid subculture (1) Aspirate the culture medium with a pipette gun, add 1-2ml of 4 ℃ organoid subculture buffer G to each well and leave for 2min. (2) Gently blow the matrigel with a pipette gun, collect it in a 15ml centrifuge tube, and let it stand at 4 °C for 10 minutes. (groups of 6-8 wells) (3) a: When the number of organoids is insufficient or the volume is small: centrifuge for 5 minutes to discard the supernatant, add an appropriate amount of organoid subculture buffer G, resuspend it and transfer it into a 1.5 ml centrifuge tube, centrifuge 300g for 5 minutes to discard the liquid for step 4. b: When the number of organoids is large or the volume is large: centrifuge for 5 minutes to discard the supernatant, add an appropriate amount of organoid subculture juice D for 2-3 minutes, add organoid subculture buffer G to terminate digestion, centrifuge for 5 minutes to discard the mixed solution, add an appropriate amount of organoid subculture buffer G to resuspend and transfer into a 1.5 ml centrifuge tube, centrifuge 300g for 5 minutes to discard the liquid for step 4. (4) After organoid collection, add matrigel for resuspension, spread 25ul of matrigel per well in a 24-well cell culture plate, and place it in an incubator for 10-15min to add 500ul of mouse normal pancreatic organoid medium A. 3. Organoid cryopreservation (1) Aspirate the culture medium with a pipette gun, add 1-2ml of 4 ℃ organoid subculture buffer G to each well and leave for 2min. (2) Gently blow the matrigel with a pipette gun, collect it in a 15ml centrifuge tube, and let it stand at 4 °C for 10 minutes. (groups of 6-8 wells) (3) Centrifuge for 5 minutes to discard the supernatant, add an appropriate amount of organoid subculture buffer G to resuspend again, and centrifuge 300g for 5 minutes to discard the liquid. (4) Add an appropriate amount of organoid cryopreservation solution F, gently blow and resuspend, take a 24-well cell culture plate as an example: the density is 2 wells for cryopreservation in 1 tube, and the volume of each tube is 1.4 ml. (5) Make good marking information, carry out program cooling, and then move it into liquid nitrogen for long-term storage. 4. Organoid resuscitation (1) Take 10ml of organoid subculture buffer G in a 15ml centrifuge tube. (2) Take out the frozen organoid cells from the liquid nitrogen tank and quickly thaw them in a 37 ℃ water bath. (3) During the water bath melting process, the cryotube needs to be gently shaken to ensure that the cryopreservation solution is completely melted within 1-2 minutes. (4) Quickly transfer the dissolved organoid cells to a 15ml centrifuge tube, gently pipette 6-8 times with a pipette, centrifuge at 300g for 5min, then remove the supernatant and collect the organoid cell pellet. Add an appropriate amount of organoid subculture buffer G, resuspend it, transfer it into a 1.5 ml centrifuge tube with 300g and centrifuge for 5 minutes. (5) Resuspend Matrigel, spread 25ul Matrigel per well in a 24-well cell culture plate, place it in an incubator for 10-15min to gel, and add 500ul mouse normal pancreatic organoid culture medium A. |
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| Synonym | Organotial Mouse Pancreatic Organoid Culture Medium Kit | ||||||||||||||||
| Description |
Kit composition:
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| Storage Temp. | Store at 4 °C, shelf life is 3 months; -20 °C, shelf life 1 year, see label for details. |
