अग्नाशय islets के मधुमेह चूहे की किडनी कैप्सूल में ट्रांसप्लांटेशन
Summary
हमारे प्रोटोकॉल के लिए स्वच्छ और आसानी से चूहों के गुर्दे कैप्सूल के तहत islets या कक्षों देने के लिए विकसित किया गया था. कक्ष अंतिम गुर्दे कैप्सूल के तहत कोशिकाओं के प्रत्यारोपण के लिए इस्तेमाल किया टयूबिंग में छर्रों में केंद्रित कर रहे हैं. इस तकनीक का आसानी कोशिकाओं और माउस के लिए तनाव को कम कर देता है.
Abstract
Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.
Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.
Protocol
Discussion
इस प्रोटोकॉल सफाई और आसानी से मधुमेह या सामान्य चूहों के गुर्दे कैप्सूल के तहत islets या कक्षों पहुंचाने के लिए एक व्यावहारिक और कुशल विकल्प प्रदान करता है. ध्यान केंद्रित और pelleting गुर्दे कैप्सूल के तहत कोशिकाओं का…
Acknowledgements
UCSF मधुमेह केंद्र. NIH UCSF DERC आइलेट कोर. JDRF. LIFESCAN इंक जॉनसन एंड जॉनसन कंपनी.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Surgical gloves | Fisher | 11-394-95(sz) | ||
| Surgical scrub sponges | Moore medical | 42940AK | ||
| Forceps | Miltex | 6-114 and 6-26 | 2 with teeth, 2 straight | |
| Dissecting scissors n=2 | Miltex | 5-290 | ||
| Oster razor (sz. 40 blade) | Fisher | 01-305-10B | ||
| 4×4 Sterile gauze pads | Moore Medical | 08252AK | ||
| Povidone Iodine pads | Moore Medical | 08486AK | ||
| Alcohol pads | Fisher | 14-819-2 | ||
| Heating pad | Moore Medical | 42508AK | ||
| 23G or 25G needles | 1″ long | |||
| Cidex solution | Moore Medical | 07535AK | for cleaning/santizing instruments | |
| PE50 polyethylene tubing | BD | 427411 | PE50, 0.965mm O.D. x 0.58mm I.D. | |
| Cidex + 28 day soln | Moore | 35625AK | ||
| Silicone tubing | Spectrum Chromat. | 123732 | 5/32”OD x 1/32” ID | |
| Instrument sterilizing container | Moore Medical | 39074AK | ||
| Straight Pipet Tips | TipOne | 1111-0810 | sterile | |
| Glass syringe w/screw-drive | Hamilton | 1001 | ||
| Cauterizing tool | Roboz | RS230 | ||
| Needle Holder | Moore Medical | 41-067 | ||
| 5-0 silk | Look | 754B | for suture (6-C) | |
| Cotton tipped swab | Moore Medical | |||
| 9mm autoclip stapler | BD | 7630 | ||
| 9mm staples | BD | 7631 | ||
| 9mm staple remover | BD | 7637 |
References
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