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Summary
Cite this Article
Copy Citation | Download CitationsSzot, G. L., Koudria, P., Bluestone, J. A. Murine Pancreatic Islet Isolation. J. Vis. Exp. (7), e255, doi:10.3791/255 (2007).
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Abstract
Protocol
Подготовка Коллагеназа
- Отвешивать коллагеназы в 50 мл конические и добавить изоляции буфера, чтобы конечная концентрация, как указано в таблице ниже. Растворите коллагеназы на вортексе.
(12 мышей + 2 дополнительных мышей = 14 мышей х 5 мл / мышь = 70 мл полное решение коллагеназы)
(70 мл х 0,5 мг / мл или 0,3 мг / мл = 35 мг или 21 мг общего коллагеназы Р)Напряжение Возраст Коллагеназа Р (мг / мл) Время (мин) C3H, Balb / С, В6 > 12 недель 0,8 17 C3H, Balb / С, В6 <= 12 недель 0,5 13 NOD, NOR, ОТСУТСТВИЯ > 12 недель 0,8 17 NOD, NOR, ОТСУТСТВИЯ <= 12 недель 0,5 17 - Внесите 2 мл коллагеназы раствора в каждую ампулу стекла и место на льду. Нагрузка каждого 5-мл шприц 3 мл коллагеназы добавить иглой 30g и место в лед (подготовка в капот).
Растяжение
- Незадолго до вскрытия, жертву животного шейки дислокации. Спрей всего тела с 70% этанола, что делает его совершенно мокрый. Сделать V-insision, начиная с области половых органов. Поверните мышь так хвост отворачивается от вас.
- Удалить кишечника в левой части открытой мыши. Это выставит поджелудочной железы и общего желчного протока.
- Место кровоостанавливающего зажима на обе стороны тонкой кишки, где желчных протоков стоков, оставляя небольшой карман для коллагеназы решение войти в кишечнике.
- Inflate поджелудочной железы через желчный проток с иглой 30g и 5 мл шприц, содержащий 3 мл холодного раствора коллагеназы, начиная с желчным пузырем.
- Удаление поджелудочной железы из тела и поместить его в силиконизированный флакон с 2 мл коллагеназы решение. Этот шаг должен быть сделан быстро и чисто, как это возможно, сведения к минимуму сбор жира и соединительной ткани.
- Место флакон на льду и повторите шаг 3 со следующей мыши.
Пищеварение
- Печать каждого флакона и поместить его в 37 ° С водяной бане. Инкубируйте 13-17 минут (время варьируется в зависимости от штамма и возраста животного).
- После истечения времени, встряхнуть флакон энергично. Поджелудочной железы должны разваливаться.
- Залить каждую переварить через большую стерильной фильтр погрузиться в стерильную 1000 мл стакан и с силой пипетки с экрана промывочным раствором. Внесите дайджесты в 50 мл пробирки conicals (3 pancreata/50 мл трубки: при п = 12, достаточно использовать промывочный буфер, чтобы сделать окончательный объем 200 мл и отказаться от ее 4x50 мл пробирки). Разбавление должно быть сделано быстро и на льду, чтобы избежать нежелательных пищеварения островок.
- Спином вниз: Пуск центрифуги. Когда она достигает 1300 оборотов в минуту, выключить его.
- Аспирируйте супернатант, оставив ~ 5 мл. Будьте очень осторожны, чтобы не аспирата гранул.
- Ресуспендируют гранул, нажав энергично рукой, а затем добавить 50 мл промывочного буфера.
- Спином вниз: Пуск центрифуги. Когда она достигает 1300 оборотов в минуту, выключить его.
- Ресуспендируют шарик и промыть 5 мл промывочного буфера.
- Передача 5 мл до 15 мл конические и спином вниз, как указано выше.
- Аспирируйте супернатант как можно более полно. (Оставшееся буфера может привести к изменению плотности Ficoll.)
Ficoll
Выполнить быстро. Долгосрочные Ficoll экспозиции является токсичным для островков.
- Убедитесь, что гранулы полностью разбит перед добавлением Ficoll (непрерывную ткань трудно ресуспендируют в Ficoll).
- Ресуспендируют гранул в 7 мл Ficoll, плотность 1,108, по вортексе энергично.
- Слой друг на плотность Ficoll 2 мл каждого из оставшихся плотности в таком порядке: 1,096, 1,069, то 1,037.
- Спиновые в течение 15 минут при 1800 оборотов в минуту на 4 градуса, с перерывом OFF!
- Выберите островки из второй слой с помощью spoid (стерильный рlastic пипетки). Передача всех коллекций в 50 мл конические, содержащей ~ 25 мл холодного буфера.
- Вымойте, как и выше, в 3 раза промывочным буфером (повторив шаг 12-15).
- Ресуспендируют островков в 20 мл RPMI-1640 (содержащей 10% FCS и пенициллин и стрептомицин, HEPES, MEM-NEAA) и аккуратно перемешать. Удалить 100 мкл образца для подсчета голосов.
- Передача 100 мкл до 35 мм грид-пластину, содержащую 1 мл среды и 1 мл дитизоном.
- Инкубируйте оставшиеся островки в RPMI 1640 году, в 37 ° C, 7% СО 2 инкубатора, в 160 мм пластин с в общей сложности 30 мл среды / пластину.
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Materials
| Name | Type | Company | Catalog Number | Comments |
| Dithizone | reagent | Dissolve 5mg Dithizone in 5 ml of DMSO in a 50 ml conical tube. Add 45 ml of normal saline and mix gently. Filter with a 60 cc syringe and a 0.22 "m syringe filter into a new 50 ml tube. Store at 4 OC, until use. Add 1 ml to each 35 mm counting Grid-plate, when needed. | ||
| PBS | Buffer | |||
| HBSS | Buffer | |||
| Isolation soln. | Buffer | HBSS containing 10 mM of HEPES, 1 mM of MgCl2, 5 mM of Glucose, pH 7.4 | ||
| Wash soln. | Buffer | Isolation buffer containing 1mM CaCl2 | ||
| Collagenase P | Boehringer Ingeheim | 1213873 | ||
| Dissection tools | Hemostat clamp (n=2), Forceps (n=2), Scissors (for skin incision an for taking out pancreas from adjacent tissue) | |||
| Syringe | sterile, 5ml, 1/mouse. | |||
| 30 G needles | sterile, 1/syringe | |||
| siliconized glass vials w/Teflon cap | Fisher Scientific | 213-018-54 | sterile, 1/mouse | |
| large sink strainer | sterile | |||
| 1000 ml Beaker | sterile | |||
| Gauze pads | 4x4, 2/mouse | |||
| plastic eye dropper | sterile | |||
| Mice | Animal | |||
Comments
58 Comments
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Where I can purchase Grid-plate?
Nunc 35mmX10mm Tissue Culture Dish, W/²mm Grid ( Sterile )1²-565-9²$²09.78500$0.4²1²$5.03Fisher800-766-7000
How did you get the densities of ficoll (1.108, 1.096, 1.069, and 1.037)?
In order to make different densities of the ficoll we use the Pollysucrose powder to prepare a stock density solution. Then it is dissolved to the desired densities.
Is there any formula to weight the exact amount of Ficoll and get the desire density? How are done the densities dilutions once you have the stock ficoll solution? Thanks, fbt
In order to make different densities of the ficoll we use the Pollysucrose powder to prepare a stock density solution. Then it is dissolved to the desired densities.
Densities are tested using: DENSITY- METER MDA 35 (Cat#: 55339) Scientific Instruments Precision machinery Electronics Anton Paar K.G. A – 8054 GRAZ. Postfach 58 Kamtner Strasse 3²²
How can you sure the degree of digestion?We use SD rat(8~10 week age) and collagenase V(sigma).Can you recommend an appropriate concentration of collagenase and digestion time for me?
Fukuoka, In the past I've isolated rat islets with ROCHE collagenase P at 1mg/mL and injected 7-10 mL into the pancreas and placed 5 ML into a 50ML glass vial or plastic conical tube.
Thanks greg szot. By the way, As I know, rat is generally expected to 400~500,and mouse ²00 islets.
How many islets do you expect or generally isolate per mouse pancreas?
We generally calculate 100-1²5 islets from older C3H mice. It could be different based on stain and age.
Can you give an estimation of what the protein concentration would be if you digested the islets? I am getting about 1ug/ul for 9pancreata... It seems low to me? Any suggestions on where I could optimize my technique? Need to do an immunoprecip & am worried about concentrations Thanks =)
I've never quantified total protien from purified isolated islets. But 1ug dŒs sound low. One cell should have about 5 pg total protein/cell. an islet has approximately 1000 cells and 9 pancreas should give you 1000 islets that should give you 5ug protien. Did you hand pick and count your purified islets? Is there a control cell-line (MIN6 mouse insulin producing cell) you can process along side the islet prep. adjusted to same cell number as the islets. Good luck.
First , I digest pancreat as you do and then,I dyed all the pancreatic mixture with trace dithizone²²90;So the islet will be become red under the microscope,which can make hand pickng easyer.But I dont know whether trace dithizone will harm the islets and efect the outcome.....
What percenage gradients match the ficoll densities that are used in your protocol? Other protocols use 11%, ²0%, ²3% and ²5% Ficoll gradients with the islets being in the ²0% layer after centrifugation. I am unsure how to make the densities used in this protocol. Thank you
We use a densitometer to determine the densities of our solutions and adjust the volumes based on density. If I was going to make 100 mL of each density and calculate percentage using a stock Ficoll with a density of 1.11² the results would be approximately: [Density 1.108x100mL]-10².36 = 8.44 = (1.11²-0.00²) – 1.0²² EC density) 0.088 95.90 mL Stock Ficoll + ² mL FBS + ².1mL Eurocollins (95.9% of a 1.11² ficoll) (note: percentages will change with different densities a stock ficoll. Example: a stock ficoll of 1.1²7 would be 8²% solution for a 1.108 final solution) [Density 1.096x100mL]-10².36 = 7.²4 = (1.11²-0.00²) – 1.0²²) 0.088 8².²7 mL Stock Ficoll + ² mL FBS + 15.73mL Eurocollins (8².²7% of a 1.11² ficoll) [Density 1.069x100mL]-10².36 = 4.54 = (1.11²-0.00²) – 1.0²²) 0.088 51.6 mL Stock Ficoll + ² mL FBS +46.4mL Eurocollins (51.6% of a 1.11² ficoll) [Density 1.037x100mL]-10².36 = 1.34 = (1.11²-0.00²) – 1.0²²) 0.088 15.² mL Stock Ficoll + ² mL FBS + 8².8mL Eurocollins (15.²% of a 1.11² ficoll)
I've never worked with Ficoll gradient. Your stock solution is made with Eurocollins? Thanks.
Eurocollins Solution Recipe (for laboratory production - 1 liter) Electrolyte Additive Solution ².09 g Potassium dihydrogen phosphate (4.18 g/² liter) 7.55 g Potassium monohydrogen phosphate (15.1 g/² liter) 1.14 g Potassium chloride (².²8 g/² liter) 0.86 g Sodium bicarbonate (1.7² g/² liter) · Dissolve and bring up to ²0 ml with distilled water (40 ml/² liter). Set a side. Glucose Solution 36.4 g Glucose, anhydrous (7².8 g/² liter) Dissolve and bring up to 1 liter with distilled water (or ² liters). Working Euro-Collins-Solution Mix 1 liter of Glucose Solution with ²0 ml of Electrolyte Solution. Sterile filter the 10²0 ml solution with a 0.²² mm stericup filter with reservoir (or ²040 ml solution into ² stericup filters). Store at 4OC
Hello, Where can I purchase Euro collins?
see comments on eurocolins above.
Thanks for your explanation, but what are the 10²,36 and 0,00² numbers: Thanks, Florencia
Hi, i have a question regarding the distension on step 5. I have tried to place the clamps on the intestine. but for some reasons the solution that i injected gŒs to the liver instead of the pancreas. Can you intrepret more about exactly where i should put the hemostat clamps on the intestine. Thanks.
Clamping the duodenum and intestine prevents your enzyme solutions from flush down into the gut and stomach it will not prevent flow into the liver. You will get enzyme in the liver, but do not worry. As your injections improve and your needle moves further down the common bile duct the tip of your needle will move past the branching to the liver and direct more enzyme into the pancreas. Be careful not to apply too much pressure to the syringe. Excessive pressure will result in bursting the intestine and pancreas inflation will suffer. Inject enzyme at a slow low pressure pace. This step is the most important because digestion quality and islet yield is dependent on the amount of enzyme injected into the pancreas.
I'm not sure how to prepare Ficoll gradient. What buffer do you use to make the stock solution? Is it Eurocollins? Thanks,
Hi, Firstly, I want to thank you for this nicely executed video particularly on a procedure that I have found hard to garner information on in the past. My first question is that in your written protocol, you have peformed calculations with 0.3 and 0.5mg/ml even though the table contains 0.5 and 0.8mg/ml collagenase solutions. I'd like to find out whether 0.3mg/ml is a viable concentration to use or if it is too low a concentration for any mouse strain? Secondly, will incubating the pancrease in collagenase solution at 37oC risk overdigestion of islets (we usually keep the inflated pancreas in a falcon tube on ice, and before incubation add warm buffer - no collagenase present). Thirdly, dŒs the sink strainer you use have a specific pore size or will any sink strainer do? We have always orders mesh with a particular pore size in the past, but if we can use these kitchen sink strainers, all the better. Cheers, Tanya (Australia)
I included a range of enzyme concentrations so that if a particular lot of enzyme is hot you would not over digest and yes, we can see digestion with a low concenration of 0.3 mg/mL. Try it some time and compare a pancreas distended with 0.0 mg/mL of collagenase. Ive never seen overdigestion at 37oC, once we've titered an enzyme lot. As for the strainer, its very large pore, it is just to grab the ducts and membrane capsule. everything else flows through.
Hi, I have a question regarding the distension: I just started to learn how to isolate pancreas, so I have a trouble to find right place for clamp - collagenase gŒs in to intestines and duodenum/stomach and everything is inflating, except the pancreas! I have tried different technique with clamping right on top of bile duct entering in to small intestines, right bellow duodenum, but again - pancreas is not inflating! Or, maybe I just can't recognize when it's inflated? Is it really visible, when pancreas is inflating? Thank you so much, Elvira
It takes some practice to correctly clamp the two sites on either side of the comon bile duct where it meets the duodenum. First, identify the duct where it meets the duodenum and then completely place a small mosquito clamp across the duodenum just to the left of the duct (near to the stomach). Do not clamp duct or the pancreas attached to the duodenum. Do the same for the right side of the duct where it meets the duodenum. Becareful not to grab the area between the two clamps with forcepts, it may casue the intestine to burst while distending the pancrease. If the stomach and small intestine distends you are not placing the clamp across the enire length of the intestine. Greg
WASEEM PAK PCMD, CAN I USE COLAGENASE 11 IN PLACE OF COLLAGENASE P FOR DIGETION CAN I ISOLATE ISLET WITHOUT DISSTINCTION OF PANCREAS
Are those siliconized glass vials w/Teflon cap from Fisher Scientific? Is the Ca#²13-018-54 right? I'm trying to order them online on the Fisher Scientific Website:however, I get "no match" from three attempts. Would you please let me know where can I get them? and how much will they cost? After incubation for 13-17 minutes (digestion) at 37c water-bath,do you shake the vial GENTLY or VIGOROUSLY? Thank you very much!
Hello, I am also having the same problem with the siliconized glass vials. Please help, thanks so much!
Gentle Shaking, let the enzyme do the work. I also checked Fisher and they are no longer carrying the vials we used, try ordering from VWR see below. The glass vials are scintillation vials (VWR 660²1-6²4) that we siliconize (Sigma; sigmacoat, SL²), wash, and autoclave. We coat the inside of the vials with ²ml of silicone and then pour to the next vial, ect.. Let the vial dry and then rinse the vial with a lot of water, because silicone is toxic to cells, the caps are placed on loosely and a piece of foil is placed over the cap and vial and they are autoclaved. We usually have 50-100 vials prepared to help save time. Scintillation vials w/ Urea Caps-Polyethylene Disc size ²4 Wheaton# 986568 VWR# 660²1-6²4 Case of 500 $²33.²3
First, I want to say how helpful this video was for me. Two questions: 1. Is the buoyancy difference due to the islet mass, or individual islet cells? In other words, if digested to individual single cells, will this phenomenon behave similarly, allowing isolation of islet and non-islet cells. ². What's the best way to make the stock ficoll: e.g. make 111.² g Ficoll powder in 100mL water for 1.11²g/mL?
Hi,
First of all, I would like to thank you for the nice detailed video protocol that you have provided on the web.
I found the protocol was very helpful and well explained; however, I still have few questions that I would appreciate if would answer:
1) Very often I have to struggle to maintain the common bile duct and the gall bladder straight because the liver tends to go underneath them. When it happens, the common bile duct bends and I end up puncturing it with the needle as I try to move it down. Do you have any suggestion to overcome this problem?
²) From how many pancreata do you get the pancreatic islets pellet, which you showed in a 50ml Falcon tube after the Ficoll at the end of the video?
Thank you for your help.
Sincerely,
Carlo
1) you need to keep your needle straight and keep injecting even if you cant fully see the injection site after entering the duct. You may want to try exposing the liver with a larger incision and you can try controling its possition with a piece of qauze.
²) I cant remember how many pancreas that were processed in the video. Normally we average 3 pancreas per tube from the pooled pancreas digest. so processing ²0 pancreas would result in 6-50mL conicals with the digest divided equally and then six ficoll tubes.
I hope this helps.
Hallo
Working in beta-cell pharmacology, I would like know if the isolation of beta cell is possible?
Hi,
That is exactly what this protocol explains about.
the process describes how to isolate and purify mouse islets. I also have a protocol that disociates the islets into individual cells. Is that what your looking for?
I will be working with rats as the source of our islets and I was curious if there is a preferential euthanasia technique for these animals. In the video cervical dislocation is stated but this is not allowed for rats according to our IACUC. Is CO² asphixiation or isoflurane anesthesia followed by exangination OK? Any recommendtions would be appreciated. And thanks for the great demonstration.
Can you culture the islets overnight and if so what medium do you recommend and is there any loss of islets due to culture?
What is the catalog number/company you use for the HBSS buffer?
At this point, I need do not need a purified islet preparation but an enriched preparation to determine if my gene of interest is expressed ot not in islet. Could you please suggest me a method I can use.
Hello!
I'm starting an insulin release assay from isolated islets using ELISA and I have some questions.
Firstly, will dithizone staining affect my ELISA results, although I will be measuring the supernatant, not iselts particularly?
Secondly, do You have some pictures of islets You have isolated, so I could be sure I'm picking the right thing?
I would appreciate Your help very much!
Hello, Yes, we have isolated the pancreatci iselt and was mesured the Insulin on the supernactant after glucose free or not Krebs ringer solution stimulation. Firstly, you must make a primary cell culture with these islets. Secondly, we quantified Insulin realising with HPLC (C18 reverse phase, isocratic tampon) method.
Zo Rakotoniaina
very good !
Some people use 11%, ²0%, ²3%, ²5% Ficoll gradient to isolate rat islets, is there any difference between the former concentrations?
Question: How to make a stock Ficoll with a density of 1.11² ?
You have mentioned "In order to make different densities of the ficoll we use the Pollysucrose powder to prepare a stock density solution. Then it is dissolved to the desired densities.
", but I donot understand it very much. Can you explain it more in detail for me? Thank you very much.
Hello there-I'm still a beginner at this procedure so i need some advice about the cannulation of the bile duct;i start from the gall bladder right?many times,especially if the animal has a lot of adipose tissue around it,the needle ends up in it...any tip for easier cannulation?
Hi!
How do you prepare the 1.11² Ficoll stock solution?
Best regards,
Fabian
Could somebody PLEASE explain how to make the Ficoll stock solution? It needs to have a density of 1.11², so you would have to dissolve ~ 31.² g of Ficoll in 100 ml. Do you have to make it in Euro-Collins as well? Do you also add FBS to the stock solution?
What are the components at the bottom of the ficoll gradient; ie in the 1.108 fraction?
Hi,
I got several questions:
1)I have used collagenase (not P type) 0.5mg/ml concentration ,Do you think it can be all right?Should i decrease the concentration?
²)I want to culture these islet,Is that possible?
Dear Greg-
It has been a while since I have made a ficoll gradient and I have some questions. First, what ficoll do you buy? 400, PM 400 or polysucrose 400? I am not sure what the difference is between the polysucrose and the ficoll? Second, when making the ficoll, I read above that you make your highest density and then the lower ones which makes sense; however I seem to recall that it takes a while to get your ficoll into solution such that you prepare it the day before, is that correct? Finally, in culturing the cells, in the video you used RPMI 1640, do you add pen/strep or FBS or anything else to the media and are the kept at 37 normal CO²? Thank you!
Lindsey, do not bother with a Ficoll gradient just use Histopaque 1077.
Just a note: Collagenase P has a very high endotoxin content which will cause a strong inflammatory response upon allo-transplantation. Roche sells Collagenase TL which is guaranteed endotoxin free and we have used it very successfully for islet isolation in mice and rats using the same incubation times as in the protocol
can i do the same experiment with wistar rat? how many days the cultured pancreatic beta cells survive? have you used these cells for any experiment? i want to use the culture for more than 7²hr. is this possible?