Murino pancreatica isolamento Islet

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Summary

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Szot, 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

Collagenasi Preparazione

  1. Pesare la collagenasi in un buffer 50 isolamento ml e aggiungere per rendere la concentrazione finale, come indicato nella tabella sottostante. Sciogliere il collagenasi vortexando.


    (12 + 2 topi topi topi in più = 14 x 5 ml / mouse = 70 ml di soluzione di collagenasi totale)


    (70 ml x 0,5 mg / ml o 0,3 mg / ml = 35 mg o 21 mg del totale collagenasi P)


    Tensione Età Collagenasi P (mg / ml) Tempo (min)
    C3H, Balb / C, B6 > 12 settimane 0,8 17
    C3H, Balb / C, B6 <= 12 settimane 0,5 13
    NOD, NOR, NON > 12 settimane 0,8 17
    NOD, NOR, NON <= 12 settimane 0,5 17


  2. Dispensare 2 ml di soluzione di collagenasi in ogni fiala di vetro e posto sul ghiaccio. Caricare ogni siringa da 5 ml con 3 ml di aghi 30g collagenasi aggiungere e mettere in ghiaccio (la preparazione in cappa).

Dilatazione

  1. Poco prima dissezione, sacrificio animale per dislocazione cervicale. Spruzzare tutto il corpo con il 70% dei ETOH, rendendolo completamente bagnato. Fare un V-Insision a partire dalla zona genitale. Ruotare il mouse in modo la coda è rivolto verso di voi.
  2. Rimuovere l'intestino verso il lato sinistro del mouse aperta. Questo esporrà il pancreas e del dotto biliare comune.
  3. Posizionare una pinza emostatica su entrambi i lati del piccolo-intestinale, dove il dotto biliare fognature, lasciando una piccola tasca per soluzione collagenasi entrare l'intestino.
  4. Gonfiare il pancreas attraverso il dotto biliare con un ago 30g e siringa da 5 ml contenente 3 ml di soluzione di collagenasi a freddo, a partire dalla cistifellea.
  5. Rimuovere il pancreas dal corpo e posizionarlo in un flaconcino siliconata contenente 2 ml di soluzione di collagenasi. Questo passo dovrebbe essere fatto rapidamente e in modo pulito possibile, riducendo al minimo la raccolta di grasso e tessuto connettivo.
  6. Posizionare il flaconcino su ghiaccio e ripetere il passo 3 con il mouse successiva.

Digestione

  1. Sigillare ogni fiala e collocarlo in un 37 ° C bagnomaria. Incubare per 13-17 minuti (il tempo varia con sforzo e l'età dell'animale).
  2. Dopo il tempo è trascorso, agitare il flacone energicamente. Il pancreas dovrebbe cadere a pezzi.
  3. Versare ogni digestione attraverso un colino grande dissipatore di sterile in un bicchiere da 1000 ml, sterile e con forza pipetta fuori dallo schermo con il tampone di lavaggio. Erogare il digerisce in 50 ml provette conicals (3 pancreata/50 tubo ml: se n = 12, basta utilizzare il tampone di lavaggio per rendere il volume finale 200 ml e dispensare a tubi 4x50 ml). La diluizione deve essere fatta rapidamente e in ghiaccio per evitare indesiderate digestione isolotto.
  4. Spin down: Avviare la centrifuga. Quando raggiunge 1300 rpm, spegnerlo.
  5. Aspirare il sopranatante, lasciando ~ 5 ml. Essere molto cauti a non aspirare il pellet.
  6. Risospendere le sfere toccando vigorosamente con la mano, poi aggiungere 50 ml di tampone di lavaggio.
  7. Spin down: Avviare la centrifuga. Quando raggiunge 1300 rpm, spegnerlo.
  8. Risospendere il pellet e lavare con 5 ml di soluzione di lavaggio.
  9. Trasferire il 5 ml a 15 ml e spin down, come sopra.
  10. Aspirare il surnatante modo più completo possibile. (Buffer rimanente potrebbe causare la variazione della densità del Ficoll).

Ficoll

Eseguire rapidamente. Esposizione a lungo termine Ficoll è tossico per gli isolotti.

  1. Assicurarsi pellet è completamente rotto prima di aggiungere Ficoll (tessuto ininterrotta è difficile da risospendere in Ficoll).
  2. Risospendere il pellet in 7 ml di Ficoll, densità 1,108, vortexando vigorosamente.
  3. Strati l'uno sopra l'densità di Ficoll 2 ml di ciascuna delle densità rimanenti in questo ordine: 1,096, 1,069, quindi 1,037.
  4. Spin per 15 minuti a 1800 rpm a 4 gradi, con pausa OFF!
  5. Scegli isolotti del secondo livello utilizzando spoid (sterili pcontagocce Lastic). Trasferire tutte le collezioni ad una conica da 50 ml contenente circa 25 ml di tampone a freddo.
  6. Lavare, come sopra, 3 volte con tampone di lavaggio (ripetendo il passaggio 12-15).
  7. Isolotti Risospendere in 20 ml di RPMI-1640 (contenente 10% FCS e la penicillina e la streptomicina, HEPES, MEM-NEAA) e mescolare delicatamente. Rimuovere 100 ul di campione per il conteggio.
  8. Trasferire 100 ul a 35 mm Griglia piastra contenente 1 ml di media e 1 ditizone ml.
  9. Incubare isolotti rimanenti in RPMI 1640, a 37 ° C, 7% di CO 2 incubatore, in 160 piastre mm con un totale di 30 ml di media / piastra.

    Figura 1

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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

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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

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