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    Rat Colon Organoid Culture

    Rat Colon Organoid Culture

    Catalog Number: abs9770 Brand: Absin
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    $476 USD
    $476 USD
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    Product Specification

    Usage 1 , original  
    （ 1 After sampling, normal rat colon tissue must be in 2-8°C Wash, prepare several petri dishes, and add 4℃ Precooled primary culture buffer for normal rat colon organoid tissue ( abs9731 ) Standby (add bispecific antibodies and gentamicin).
    （ 2 The tissue was placed in a petri dish, washed three times with primary culture buffer for normal rat colon organoid tissue, and impurities were removed. The intestinal mucosa of the rat colon tissue was then removed using ophthalmic scissors or a scalpel, and the tissue was cut into a volume of approximately... 10-30mm3 Tissue blocks.
    （ 3 Primary tissue digestive fluid for tissue use in normal rat colon tissue ( 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 A small amount of liquid was taken and observed under a microscope. More crypts were observed under the microscope. Three times the volume of primary culture buffer for normal rat colon organoid tissue was added to terminate the digestion.
    （ 5 ) 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 The supernatant was then removed, and the primary culture buffer of normal rat colon organoid tissue was added for resuspension and centrifugation.
    （ 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. 25μL Tissue matrix glue mixture for board laying ( 4℃ (Next operation).
    （ 8 Place the prepared culture plate in... 37℃ Incubator 10-15min Gelatinization, addition of rat normal colon organoid medium (restored to room temperature) for culture.
    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, add an appropriate amount of rat normal colon organoid subculture buffer, resuspend and transfer. 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 digestive fluid ( abs9520 Digestion 2-3min Add normal rat colon organoid subculture buffer to terminate digestion and centrifuge. 5min The mixture was discarded, and an appropriate amount of rat normal colon organoid subculture buffer was added for resuspension and transfer. 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. 25μL Matrix glue is laid on 24 In well cell culture plates, place in an incubator. 10-15min Add 500μL Organoid medium for normal colon in rats.
    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 Discarding the supernatant, adding an appropriate amount of rat normal colon organoid subculture buffer to resuspend 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 10mL Rat 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 an appropriate amount of rat normal colon organoid subculture buffer for resuspension and transfer. 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 Organoid medium for normal colon in rats.
    Synonym Rat Colon Organoid Culture
    Storage Temp. Store at -20°C with a shelf life of 12 months.