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    Fetal Rat Small Intestine Organoid Culture

    Fetal Rat Small Intestine Organoid Culture

    Catalog Number: abs9767 Brand: Absin
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    $476 USD
    $476 USD
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    Product Specification

    Usage 1 , original  
    （ 1 The normal small intestine tissue of rat fetuses after sampling must be in 2-8°C Wash, prepare several petri dishes, and add 4℃ Precooled primary culture buffer for normal small intestine organoids in rat fetuses #abs9731 Standby (added bispecific antibody and gentamicin).
    （ 2 The tissue was placed in a petri dish, washed three times with primary culture buffer for normal rat fetal small intestinal organoid tissue, impurities were removed, the intestinal mucosa of the fetal rat small intestinal tissue was removed with ophthalmic scissors or a scalpel, and the tissue was cut into a volume of approximately... 10-30mm3 Tissue blocks.
    （ 3 Digestive solution of primary tissue of normal small intestine tissue of rat fetus for tissue use #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 small intestinal organoid tissues of rat fetuses was added to terminate 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 Then the supernatant was removed, and the primary culture buffer of normal small intestine organoid tissue of rat fetus was added to resuspend and centrifuge.
    （ 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 Gelatinization was performed, and normal small intestine organoid culture medium of rat fetuses was added (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 normal small intestine organoid subculture buffer for rat fetuses, and 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 digestion solution. #abs9520 digestion 2-3min Add subculture buffer for organoids from the normal small intestine of rats and fetuses to terminate digestion and centrifuge. 5min Discard the mixture, add an appropriate amount of normal small intestine organoid subculture buffer for rat fetuses, and resuspend 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. 25ul Matrix glue is laid on 24 In well cell culture plates, place in an incubator. 10-15min Add 500ul Organoid culture medium in the normal small intestine of the rat fetus.
    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 fetal rat normal small intestine organoid subculture buffer for resuspension, 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 fetal rat normal small intestine 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 organoid subculture buffer for normal small intestine of rat fetuses for resuspension and transfer 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 500ul Organoid culture medium in the normal small intestine of the rat fetus.
    Synonym Fetal Rat Small Intestine Organoid Culture
    Storage Temp. Store at -20°C with a shelf life of 1 year.