Product Specification| Usage | 1. Primary
(1) The normal colon tissue of mice after collection 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 stem 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) Disinfect the sampling bottle, put the tissue in a petri dish, wash it three times with primary culture buffer B, remove impurities, and remove the intestinal mucosa of colon tissue with ophthalmic scissors or scalpel, and cut it into a volume of about 1-3mm3The tissue block.
(4) The tissues were digested with digestive juice C of normal colon primary tissue of mice, and digested by shaking at 4 ℃ for 10 minutes (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 colon 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 organ collection, add Matrigel for resuspension, spread 25ul of Matrigel per well in a 24-well cell culture plate, place it in an incubator for 10-15min, and add 500ul of mouse normal colon 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 colon organoid medium A. | | Description | Kit composition:
| Component Name | Specifications | | Mouse normal colon organoid medium A | 100ml | | Primary culture buffer B | 250ml | | Mouse normal colon primary tissue digestive juice C | 30ml | | Organoid passage digestive juice D | 30ml | | Tissue preservation solution E | 100ml | | Organoid Cryopreservation Solution F | 20ml | | Organoid Subculture Buffer G | 250ml |
| | Storage Temp. | Stored at 4 ℃, valid for 3 months; Store at-20 ℃, shelf life is 1 year, see label for details. |
bio-equip.cn AntBio is a biotechnology group company dedicated to serving life sciences, aiming to help scientists accelerate research and improve work efficiency. AntBio provides comprehensive and high-quality reagent tools for basic research, drug development, and diagnosis, including research grade antibodies, proteins, biochemical reagents, and assay kits. These research tools are widely used in different segments of life science research. The group company currently consists of three brands, Absin, Starter-Bio and UA-Bio.
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