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    Organotial Mouse Normal Colon Organoid Culture Kit

    Organotial Mouse Normal Colon Organoid Culture Kit

    Catalog Number: abs9985 Brand: Absin
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    $1,032 USD
    $1,032 USD
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    Product Details

    Product Specification

    Usage 1 , Original generation:
    （ 1 The normal colon tissue of the mice after sampling must be placed in a precooled ( 2-8°C Tissue preservation solution E The sample bottles were quickly transported to a clean laboratory for tissue processing and stem cell separation, photographed, and information recorded.
    （ 2 Prepare several petri dishes and add... 4℃ Precooled primary culture buffer B Backup.
    （ 3 The sample bottles were sterilized, and the tissues were placed in petri dishes using primary culture buffer. B After cleaning three times, remove impurities. Use ophthalmic scissors or a scalpel to remove the intestinal mucosa of the colon tissue and cut it into a volume of approximately... 1-3mm3 Tissue blocks.
    （ 4 Digestive fluid from primary tissue of normal colon in mice for tissue use C digestion, 4℃ oscillation digestion 10min (Observe the digestion situation at any time during the digestion process).
    （ 5 Take a small amount of liquid and observe it under a microscope. A large number of individual cells or 70μm After the following cell clusters, add three times the volume of primary culture buffer B Termination of digestion.
    （ 6 ) Use 100μm The filtrate is filtered using a sieve with a certain pore size, and the filtrate is collected and then... 300g enrichment centrifugation 5min Then remove the supernatant and add primary culture buffer B Resuspend and centrifuge.
    （ 7 Matrix rubber calculation: No. 6 After the step, observe the collected tissue volume and add 25 Matrix glue with twice the tissue volume ( abs9495 Resuspend the slab.
    （ 8 ） 24 Taking well cell culture plates as an example, each well is dispensed with gel. 25μL Tissue matrix glue mixture for board laying ( 4℃ (Next operation).
    （ 9 Place the prepared culture plate in... 37℃ Incubator 10-15min Gelatinization, addition of mouse normal colon organoid medium A (Return to room temperature) for incubation.
    2 Organoid subculture:
    （ 1 Remove the culture medium with a pipette and add to each well. 1-2mL 4℃ organoid subculture buffer G 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 of organoid subculture buffer G Resuspend and move in 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. D digestion 2-3min Add organoid subculture buffer G Terminate digestion and centrifuge 5min Discard the mixture and add an appropriate amount of organoid subculture buffer G Resuspend and move in 1.5mL centrifuge tube, 300g centrifuge 5min Discard the liquid for the first 4 step.
    （ 4 After organ collection, matrix gel was added for resuspension, and each well... 25μL Matrix glue is laid on 24 In well cell culture plates, place in an incubator. 10-15min Add 500μL mouse normal colon organoid culture medium A 。
    3 Organoid cryopreservation:
    （ 1 Remove the culture medium with a pipette and add to each well. 1-2mL 4℃ organoid subculture buffer G 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 of organoid subculture buffer G Hanging again, 300g centrifuge 5min Discard the liquid.
    （ 4 Add an appropriate amount of organoid cryopreservation solution. F Gently blowing and resuspending, 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 10ml organoid subculture buffer G At 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 an appropriate amount of organoid subculture buffer G Resuspend and move in 1.5mL centrifuge tube 300g centrifuge 5min 。
    （ 5 Resuspend the matrix gel, per well 25μL Matrix glue is laid on 24 In well cell culture plates, place in an incubator. 10-15min Gelation, addition 500μL mouse normal colon organoid culture medium A 。
    Description Kit Composition:
    Component Name Specification
    mouse normal colon organoid culture medium A 100mL
    primary culture buffer B 250mL
    Digestive fluid of primary tissue of normal colon in mice C 30mL
    organoid passage digestive fluid D 30mL
    tissue preservation solution E 100mL
    organoid cryopreservation fluid F 20mL
    organoid subculture buffer G 250mL
    Component Kit Composition:
    Component Name Specification
    mouse normal colon organoid culture medium A 100ml
    primary culture buffer B 250ml
    Digestive fluid of primary tissue of normal colon in mice C 30ml
    organoid passage digestive fluid D 30ml
    tissue preservation solution E 100ml
    organoid cryopreservation fluid F 20ml
    organoid subculture buffer G 250ml
    Storage Temp. Store at -20℃ for 12 months.