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 does 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  dithizone。So the islet will be become red under the

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)

1. 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.

1. Glucose Solution

36.4 g     Glucose, anhydrous (72.8 g/2 liter)

1.  
   • Dissolve and bring up to 1 liter with distilled water (or 2 liters).

1. 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 4^OC

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 goes 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, does 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 goes 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?