| Usage | 1. Primary
(1) the normal colonic tissue of balb/c mice after sampling must be cleaned at 2-8 ℃, several Petri dishes should be prepared, and primary culture buffer B pre cooled at 4 ℃ should be added for standby (double antibody and gentamicin should be added)
(2) put the tissue into the Petri dish, wash it three times with primary culture buffer B, remove impurities, remove the intestinal mucosa of Balb/c mouse colon tissue with ophthalmic scissors or scalpel, and cut it into a tissue block with a volume of about 10-30mm3
(3) the tissues were digested with the digestive juice C of the primary normal colon tissues of balb/c mice, with shaking digestion at 4 ℃ for 20-25min or standing digestion for 40-50min (observe the digestion at any time during the digestion process)
(4) take a small amount of liquid and observe under the microscope. More crypts are observed under the microscope. Add three times the volume of primary culture buffer B to stop digestion
(5) filter with a 100um pore size screen, collect the filtrate, remove the supernatant after centrifugation at 300g for 5min, add primary culture buffer B and resuspend for centrifugation
(6) Matrigel calculation: after step 5, observe the collected tissue volume, add 25 times the tissue volume of Matrigel (abs9495) to resuspend the plate
(7) take a 24 well cell culture plate as an example, dispense 25ul of tissue Matrigel mixture per well for plating (operate at 4 ℃)
(8) put the laid culture plate into a 37 ℃ incubator for 10-15min to form a gel, and add balb/c mouse normal colon organoid medium a (restore room temperature) for culture
2. Organoid subculture
(1) pipette the culture medium, add 1-2ml of 4 ℃ organoid subculture buffer g to each well and place it for 2min
(2) pipette the Matrigel gently, collect it in a 15ml centrifuge tube, and let it stand at 4 ℃ for 10min. (each 6-8 well is a group)
(3) a: the number of organoids is insufficient or the volume is small: centrifuge for 5min to discard the supernatant, add an appropriate amount of organoid subculture buffer g to resuspend and transfer into a 1.5ml centrifuge tube, centrifuge for 5min at 300g to discard the liquid for step 4
b: When the number of organoids is large or the volume is large: centrifuge for 5min to discard the supernatant, add an appropriate amount of organoid passage digest D for digestion for 2-3min, add organoid passage culture buffer g to stop digestion, centrifuge for 5min to discard the mixture, add an appropriate amount of organoid passage culture buffer g to resuspend and transfer into a 1.5ml centrifuge tube, centrifuge for 5min at 300g to discard the liquid for step 4
(4) after organoid collection, add Matrigel to resuspend, lay 25ul Matrigel per well in a 24 well cell culture plate, place in the incubator for 10-15min, and add 500ul of balb/c mouse normal colon organoid culture medium A
3. Organoid cryopreservation
(1) pipette the culture medium, add 1-2ml of 4 ℃ organoid subculture buffer g to each well and place it for 2min
(2) pipette the Matrigel gently, collect it in a 15ml centrifuge tube, and let it stand at 4 ℃ for 10min. (each 6-8 well is a group)
(3) centrifuge for 5min to discard the supernatant, add an appropriate amount of organoid subculture buffer g to resuspend, and centrifuge for 5min at 300g 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 and 1 tube is frozen, with a volume of 1.4ml per tube
(5) mark the information, and after cooling down the program, transfer it to liquid nitrogen for long-term storage
4. Organoid resuscitation
(1) take 10ml of organoid subculture buffer g and put it into a 15ml centrifuge tube
(2) take out the frozen organoid cells from the liquid nitrogen tank and quickly put them into a 37 ℃ water bath pot for melting
(3) during the water bath thawing process, it is necessary to gently shake the freezing tube to ensure that the frozen solution is completely thawed in 1-2min
(4) quickly transfer the dissolved organoid cells to a 15ml centrifuge tube, gently blow 6-8 times with a pipette, centrifuge at 300g for 5min, and then remove the supernatant and collect the organoid cell pellet. Add an appropriate amount of organoid subculture buffer g to resuspend and transfer into a 1.5ml centrifuge tube. Centrifuge for 5min at 300g
(5) resuspend Matrigel, lay 25ul Matrigel per well in a 24 well cell culture plate, place in an incubator for 10-15min to form a gel, and add 500ul of balb/c mouse normal colon organoid medium A. |
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