小鼠胰岛隔离

Biology

Your institution must subscribe to JoVE's Biology section to access this content.

Fill out the form below to receive a free trial or learn more about access:

 

Summary

Cite this Article

Copy Citation | Download Citations

Szot, G. L., Koudria, P., Bluestone, J. A. Murine Pancreatic Islet Isolation. J. Vis. Exp. (7), e255, doi:10.3791/255 (2007).

Please note that all translations are automatically generated.

Click here for the english version. For other languages click here.

Abstract

Protocol

胶原酶制备

  1. 称取50 ml锥形加隔离缓冲 ,使终浓度,如表所示的胶原酶。涡旋溶解胶原酶。


    (+ 2额外的小鼠12只小鼠= 14小鼠× 5毫升/鼠标= 70毫升的总胶原酶溶液)


    (70毫升× 0.5毫克/毫升或0.3 mg / ml的胶原酶P总= 35毫克或21毫克)


    应变年龄胶原酶P(mg / ml的) 时间(min)
    C3H,BALB / C,B6 > 12周 0.8 17
    C3H,BALB / C,B6 <= 12周 0.5 13
    点头,NOR,非 > 12周 0.8 17
    点头,NOR,非 <= 12周 0.5 17


  2. 免除到每个玻璃小瓶,置于冰上胶原酶溶液2毫升。每5毫升注射器装入3毫升胶原酶添加30克针和冰的地方准备在引擎盖。

腹胀

  1. 前清扫术,颈椎脱位而牺牲的动物。喷雾用70%的ETOH全身,使其完全湿透。请在生殖器部位开始V型insision。所以尾巴正面临着远离你的鼠标旋转。
  2. 删除打开鼠标左侧肠。这将会使胰腺和胆总管。
  3. 广场上的小肠,胆管水渠两侧的止血钳,留下一个小口袋胶原酶溶液进入肠道。
  4. 膨胀通过与30克针和包含3毫升冷胶原酶溶液5毫升注射器的胆管,胰腺,胆囊开始。
  5. 从体内取出的胰腺和它放置在一个硅的含胶原酶溶液2毫升的小瓶。这一步应该很快完成,并且尽可能干净,减少脂肪和结缔组织的集合。
  6. 放置在冰上,然后重复步骤3与下鼠标的小瓶。

消化

  1. 每个小瓶密封,放入37℃水浴。孵育13-17分钟(时间与应变和动物的年龄而异)。
  2. 时间结束后,大力摇动小瓶。胰腺土崩瓦解。
  3. 通过大型无菌水槽滤网倒入每个消化成无菌的1000毫升烧杯中,并有力地移液器关闭屏幕,用洗涤液。分送消化到50毫升conicals管(3 pancreata/50 ml管:如果n = 12,使用足够的洗涤液,使终体积为200毫升和分配到4x50毫升管)。稀释应采取迅速和冰 ,以避免不必要的胰岛消化
  4. 离心:启动离心机。当它达到1300转时,将其关闭。
  5. 吸出上清液,留下〜5毫升。必须非常谨慎,不吸沉淀。
  6. 用你的手大力攻悬浮颗粒,然后添加50毫升缓冲液洗。
  7. 离心:启动离心机。当它达到1300转时,将其关闭。
  8. 5毫升洗涤液,重悬沉淀和洗涤。
  9. 转让5毫升到15毫升锥形和自旋向下,如上。
  10. 尽可能完全吸出上清液。 (剩余的缓冲区可能导致聚蔗糖密度的变化。)

聚蔗糖

快速执行。长期聚蔗糖接触有毒的小岛。

  1. 确保颗粒是彻底打破了之前加入聚蔗​​糖(完整的组织是很难重悬在聚蔗糖)。
  2. 涡旋大力7毫升的聚蔗糖,密度1.108,重悬沉淀。
  3. 层上的每个聚蔗糖2毫升为了在这余下的密度密度:1.096,1.069,那么1.037。
  4. 4度在1800 rpm的旋转15分钟, 以断绝!
  5. 选择从第二层使用spoid(无菌p胰岛lastic吸管)。将所有集合的50毫升锥形含有约25毫升冷缓冲区。
  6. 以上,洗净,用洗涤液(重复步骤12-15)的3倍。
  7. 悬浮胰岛在20毫升的RPMI - 1640(含10%胎牛血清和青霉素和链霉素,HEPES的MEM - NEAA),轻轻混匀。取出100 UL的样品进行计数。
  8. 转移100 UL 35毫米网格板含1毫升媒体和1毫升双硫腙。
  9. 的RPMI 1640培养尚存的胰岛细胞,在37 ° C,7%的CO 2培养箱,在160毫米板共30毫升媒体/板。

    图1

Subscription Required. Please recommend JoVE to your librarian.

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

DOWNLOAD MATERIALS LIST

Comments

58 Comments

  1. Where I can purchase Grid-plate? 

    Reply
    Posted by: Anonymous
    March 18, 2008 - 5:21 PM
  2. Nunc 35mmX10mm Tissue Culture Dish, W/²mm Grid ( Sterile )1²-565-9²$²09.78500$0.4²1²$5.03Fisher800-766-7000

    Reply
    Posted by: Anonymous
    March 25, 2008 - 11:59 AM
  3. Reply
    Posted by: Anonymous
    March 18, 2008 - 5:22 PM
  4. How did you get the densities of ficoll (1.108, 1.096, 1.069, and 1.037)?

    Reply
    Posted by: Anonymous
    May 23, 2008 - 2:35 PM
  5. 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.

    Reply
    Posted by: Anonymous
    June 9, 2008 - 3:30 PM
  6. 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

    Reply
    Posted by: Florencia B.
    March 31, 2009 - 10:01 AM
  7. 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.

    Reply
    Posted by: Anonymous
    June 9, 2008 - 3:31 PM
  8. 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

    Reply
    Posted by: Anonymous
    June 9, 2008 - 6:54 PM
  9. 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?

    Reply
    Posted by: Anonymous
    September 21, 2008 - 9:00 AM
  10. 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.

    Reply
    Posted by: Anonymous
    October 8, 2008 - 3:57 PM
  11. Thanks greg szot. By the way, As I know, rat is generally expected to 400~500,and mouse ²00 islets.

    Reply
    Posted by: Anonymous
    October 10, 2008 - 8:38 PM
  12. How many islets do you expect or generally isolate per mouse pancreas?

    Reply
    Posted by: Anonymous
    October 10, 2008 - 2:03 PM
  13. We generally calculate 100-1²5 islets from older C3H mice.  It could be different based on stain and age.

    Reply
    Posted by: Anonymous
    October 17, 2008 - 4:48 PM
  14. 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 =)

    Reply
    Posted by: Anonymous
    October 16, 2008 - 9:57 PM
  15. 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.

    Reply
    Posted by: Anonymous
    October 17, 2008 - 4:45 PM
  16. 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.....

    Reply
    Posted by: Anonymous
    October 17, 2008 - 8:53 PM
  17. 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

    Reply
    Posted by: Anonymous
    October 20, 2008 - 12:39 PM
  18. 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)

    Reply
    Posted by: Anonymous
    October 23, 2008 - 1:37 PM
  19.  I've never worked with Ficoll gradient.  Your stock solution is made with Eurocollins? Thanks.

    Reply
    Posted by: Anonymous
    November 21, 2008 - 2:04 PM
  20. 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  

    Reply
    Posted by: Anonymous
    March 30, 2009 - 3:06 PM
  21. Hello, Where can I purchase Euro collins?

    Reply
    Posted by: Anonymous
    March 20, 2009 - 7:12 PM
  22. see comments on eurocolins above.

    Reply
    Posted by: Anonymous
    March 30, 2009 - 3:07 PM
  23. Thanks for your explanation, but what are the 10²,36 and 0,00² numbers: Thanks, Florencia

    Reply
    Posted by: Florencia B.
    March 30, 2009 - 11:45 AM
  24. 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.

    Reply
    Posted by: Anonymous
    October 23, 2008 - 12:26 PM
  25. 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.

    Reply
    Posted by: Anonymous
    October 23, 2008 - 1:46 PM
  26. I'm not sure how to prepare Ficoll gradient.  What buffer do you use to make the stock solution?  Is it Eurocollins? Thanks,

    Reply
    Posted by: Anonymous
    November 21, 2008 - 2:07 PM
  27. 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)  

    Reply
    Posted by: Anonymous
    November 29, 2008 - 8:11 PM
  28. 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.

    Reply
    Posted by: Anonymous
    March 30, 2009 - 3:03 PM
  29. 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

    Reply
    Posted by: Anonymous
    December 18, 2008 - 12:01 PM
  30. 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

    Reply
    Posted by: Anonymous
    March 30, 2009 - 2:52 PM
  31. WASEEM PAK PCMD, CAN I USE COLAGENASE 11 IN PLACE OF COLLAGENASE P FOR DIGETION CAN I ISOLATE ISLET WITHOUT DISSTINCTION OF PANCREAS

    Reply
    Posted by: Anonymous
    February 4, 2009 - 2:13 AM
  32. 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!

    Reply
    Posted by: Anonymous
    February 18, 2009 - 4:18 PM
  33. Hello, I am also having the same problem with the siliconized glass vials. Please help, thanks so much!

    Reply
    Posted by: Anonymous
    February 23, 2009 - 3:11 PM
  34. 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 

    Reply
    Posted by: Anonymous
    March 30, 2009 - 3:09 PM
  35. 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?

    Reply
    Posted by: Anonymous
    April 13, 2009 - 8:48 PM
  36. 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

    Reply
    Posted by: Anonymous
    May 28, 2009 - 5:03 AM
  37. 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.

    Reply
    Posted by: Anonymous
    June 17, 2009 - 7:44 PM
  38. Hallo
    Working in beta-cell pharmacology, I would like know if the isolation of beta cell is possible?

    Reply
    Posted by: Zo R.
    June 11, 2009 - 5:24 AM
  39. Hi,
    That is exactly what this protocol explains about.

    Reply
    Posted by: Anonymous
    June 11, 2009 - 12:47 PM
  40. 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?

    Reply
    Posted by: Anonymous
    June 17, 2009 - 7:37 PM
  41. 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.

    Reply
    Posted by: Anonymous
    July 7, 2009 - 12:16 PM
  42. Can you culture the islets overnight and if so what medium do you recommend and is there any loss of islets due to culture?

    Reply
    Posted by: Anonymous
    August 8, 2009 - 9:42 AM
  43. What is the catalog number/company you use for the HBSS buffer?

    Reply
    Posted by: Anonymous
    August 25, 2009 - 12:35 PM
  44. 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.

    Reply
    Posted by: Anonymous
    September 14, 2009 - 11:53 AM
  45. 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!

    Reply
    Posted by: M. I.
    November 23, 2009 - 11:36 AM
  46. 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

    Reply
    Posted by: Anonymous
    November 29, 2009 - 10:39 PM
  47. very good !

    Reply
    Posted by: Anonymous
    December 23, 2009 - 1:16 PM
  48. Some people use 11%, ²0%, ²3%, ²5% Ficoll gradient to isolate rat islets, is there any difference between the former concentrations?

    Reply
    Posted by: Anonymous
    January 24, 2010 - 8:04 AM
  49. 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.

    Reply
    Posted by: ling x.
    March 26, 2010 - 6:50 PM
  50. 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?

    Reply
    Posted by: Anonymous
    May 12, 2010 - 10:52 PM
  51. Hi!

    How do you prepare the 1.11² Ficoll stock solution?

    Best regards,
    Fabian

    Reply
    Posted by: Anonymous
    October 4, 2010 - 10:47 AM
  52. 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?

    Reply
    Posted by: Anonymous
    October 16, 2010 - 1:52 PM
  53. What are the components at the bottom of the ficoll gradient; ie in the 1.108 fraction?

    Reply
    Posted by: Anonymous
    April 14, 2011 - 9:07 AM
  54. 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?

    Reply
    Posted by: Anonymous
    May 31, 2011 - 7:24 AM
  55. 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!

    Reply
    Posted by: Anonymous
    July 21, 2011 - 5:13 PM
  56. Lindsey, do not bother with a Ficoll gradient just use Histopaque 1077.

    Reply
    Posted by: Anonymous
    July 23, 2011 - 9:31 PM
  57. 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

    Reply
    Posted by: Anonymous
    September 13, 2011 - 1:00 PM
  58. 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?

    Reply
    Posted by: Piyush C.
    September 27, 2011 - 3:23 AM

Post a Question / Comment / Request

You must be signed in to post a comment. Please or create an account.

Usage Statistics