Organotial人肾癌类器官培养试剂盒
Organotial人肾癌类器官培养试剂盒
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Product Specification
| Usage |
1 , original （ 1 Organizational recommendations after data collection are as follows: 2-8°C Stored under conditions, quickly transported to a clean laboratory for tissue processing and 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 three cleanings, the tissue is cut into a volume of approximately... using ophthalmic scissors or a scalpel. 1-3mm3 Tissue blocks. （ 4 Human renal cancer primary tissue digestive fluid for tissue use C digestion, 37℃ oscillation digestion 15-25min (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 70um After the following cell clusters, add three times the volume of primary culture buffer B Termination of digestion. （ 6 ) 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 remove the supernatant and add primary culture buffer B Re-hang, 300g enrichment centrifugation 5min Move back to Shangqing. （ 7 The cell pellet contained red blood cells. Discard the supernatant and add... 1-2mL erythrocyte lysate 1-2min Then, dilute to 10mL ， 300g centrifuge 5min 。 （ 8 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. （ 9 ） 24 Taking well cell culture plates as an example, each well is dispensed with gel. 25uL-30uL Tissue matrix glue mixture for board laying ( Note: Matrix adhesive maintained 0-4℃ (Next operation). （ 10 Place the prepared culture plate in... 37℃ Incubator 10-15min After the matrix gum sets, add... 500-750uL human renal cancer organoid culture 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 10-15min 。 (each 6-8 (The holes are a group) （ 3 ） a : Insufficient number or small volume of organoids: 300g centrifuge 5min Discard the supernatant and add 1mL 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 of organoids is large or the volume is large: 300g centrifuge 5min Discard the supernatant and add 1-2mL organoid passage digestive fluid D digestion 2-4min , add 3 Organoid subculture buffer with twice the volume of organoid digestive fluid G Termination of digestion, 300g 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 Abandon the Shangqing and proceed with 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 。 After the matrix gum sets, add to each well 500-750uL human renal cancer 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 ) each 3 Hole addition 2mL organoid cryopreservation fluid F Gently blow and mix well, then transfer to cell cryopreservation tubes. Each tube... 1mL 。 （ 5 After marking the information, performing program cooling, move to... -80°C In the refrigerator, 48h Then, it was transferred to liquid nitrogen for long-term storage. Or put in 4℃ refrigerator 40min Then, put in -20℃ in the refrigerator 2h , move to -80℃ In the refrigerator, 48h Afterwards, it was placed in a liquid nitrogen tank for 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 should be gently shaken to ensure that the cryopreservation solution is completely thawed in a short time. （ 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, move in 1.5mL centrifuge tube 300g centrifuge 5min They abandoned the Shangqing （ 5 ) Add per cryopreservation tube 100ul Matrix gel resuspension, per well 25uL Matrix glue is laid on 24 In well cell culture plates, place in an incubator. 10-15min Gelation, addition 500-750uL human renal cancer organoid culture medium A 。 |
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| Description |
Kit Composition:
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| Storage Temp. | Store at -20℃ for 1 year. |
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Immunohistochemistry
