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    Mouse(balb/C) Colon Organoid Culture

    Mouse(balb/C) Colon Organoid Culture

    Catalog Number: abs9765 Brand: Absin
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    $476 USD
    $476 USD
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    Product Details

    Product Specification

    Usage 1 , original  
    （ 1 ) After sampling Balb/C Normal colon tissue in mice must be 2-8°C Wash, prepare several petri dishes, and add 4℃ pre-cooled Balb/C Mouse normal colon organoid tissue primary culture buffer #abs9731 Standby (added bispecific antibody and gentamicin).
    （ 2 Place the tissue in a petri dish and utilize... Balb/C After washing the primary culture buffer of normal colonic organoid tissue in mice three times, remove impurities, and then use ophthalmic scissors or a scalpel to... Balb/C The intestinal mucosa of mouse colon tissue was removed, and the tissue was cut into a volume of approximately [size missing] using ophthalmic scissors or scalpels. 10-30mm3 Tissue blocks.
    （ 3 ) Organizational Balb/C Primary tissue digestive fluid of normal colon tissue in mice #abs9644 digestion, 4℃ oscillation digestion 20-25min Or allow to stand and digest. 40-50min (Observe the digestion situation at any time during the digestion process).
    （ 4 Take a small amount of liquid and observe it under a microscope. If more crypts are observed under the microscope, add three times the volume. Balb/C Mouse normal colon organoid tissue primary culture buffer terminated digestion.
    （ 5 ) Use 100um The filtrate is filtered using a sieve with a certain pore size, and the filtrate is collected and then... 300g enrichment centrifugation 5min Move back to remove the supernatant and add Balb/C Mouse normal colon organoid tissue primary culture buffer was resuspended and centrifuged.
    （ 6 Matrix rubber calculation: No. 5 After the step, observe the collected tissue volume and add 25 double tissue volume matrix glue #abs9495 Re-hang the slab.
    （ 7 ） 24 Taking well cell culture plates as an example, each well is dispensed with gel. 25ul Tissue matrix glue mixture for board laying ( 4℃ (Next operation).
    （ 8 Place the prepared culture plate in... 37℃ Incubator 10-15min Gelation, addition Balb/C Mice were cultured in normal colon organoid medium (restored to room temperature).
    2 Organoid subculture
    （ 1 Remove the culture medium with a pipette and add to each well. 1-2ml 4℃ organoid subculture buffer #abs9730 placement 2min 。
    （ 2 Gently blow the matrix glue with a pipette to collect... 15ml In the centrifuge tube, 4℃ standing 10min 。 (each 6-8 (The holes are a group)
    （ 3 ） a : Insufficient number or small volume of organoids: centrifuge 5min Discard the supernatant and add an appropriate amount. Balb/C Resuspension and transfer of normal colon organoid subculture buffer in mice 1.5ml centrifuge tube, 300g centrifuge 5min Discard the liquid for the first 4 step.
             b When the number or volume of organoids is large: Centrifuge 5min Discard the supernatant and add an appropriate amount of organoid passage digestion solution. #abs9520 digestion 2-3min , add Balb/C Digestion was terminated in mouse normal colon organoid subculture buffer and centrifuged. 5min Discard the mixture and add an appropriate amount. Balb/C Resuspension and transfer of normal colon organoid subculture buffer in mice 1.5ml centrifuge tube, 300g centrifuge 5min Discard the liquid for the first 4 step.
    （ 4 After organoids were collected, matrix gel was added for resuspension, and each well was resuspended. 25ul Matrix glue is laid on 24 In well cell culture plates, place in an incubator. 10-15min Add 500ulBalb/C Mouse normal colon organoid medium.
    3 Organoid cryopreservation
    （ 1 Remove the culture medium with a pipette and add to each well. 1-2ml 4℃ organoid subculture buffer placement 2min 。
    （ 2 Gently blow the matrix glue with a pipette to collect... 15ml In the centrifuge tube, 4℃ standing 10min 。 (each 6-8 (The holes are a group)
    （ 3 Centrifugation 5min Discard the supernatant and add an appropriate amount. Balb/C The mouse normal colon organoid subculture buffer was resuspended again, 300g centrifuge 5min Discard the liquid.
    （ 4 Add an appropriate amount of organoid cryopreservation solution, gently blow and resuspend, and 24 Taking well cell culture plates as an example: the density is 2 Individual hole cryopreservation 1 Tubes, volume per tube 1.4ml 。
    （ 5 Mark the information, cool it down procedurally, and then transfer it to liquid nitrogen for long-term storage.
    4 Organoid resuscitation
    （ 1 ) take 10mlBalb/C Mouse normal colon organoid subculture buffer 15ml In the centrifuge tube.
    （ 2 Remove the frozen organoid cells from the liquid nitrogen tank and quickly place them in... 37℃ Melt in a water bath.
    （ 3 During the water bath thawing process, the freezing tube needs to be gently shaken to ensure that the cryopreservation solution is... 1-2min It completely dissolves inside.
    （ 4 ) Rapidly transfer the lysed organoid cells to 15ml Centrifuge tube, gently blow with a pipette 6-8 next time, 300g centrifuge 5min The supernatant is then removed and the organoid cell pellet is collected. Add appropriate amount Balb/C Resuspension and transfer of normal colon organoid subculture buffer in mice 1.5ml centrifuge tube 300g centrifuge 5min 。
    （ 5 Resuspend the matrix gel, per well 25ul Matrix glue is laid on 24 In well cell culture plates, place in an incubator. 10-15min Gelation, addition 500ulBalb/C Mouse normal colon organoid medium.
    Synonym Mouse(balb/C) Colon Organoid Culture
    Storage Temp. Store at -20°C with a shelf life of 1 year.