Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Cell Biology and Cell Culture
Register Search Today's Posts Mark Forums Read

Cell Biology and Cell Culture Cell Biology Forum. Cell Culture Forum. Post and ask questions about cell culturing, cell lysis, cell transfection, cell growth, and cell biology.


mouse hepatocytes isolation

mouse hepatocytes isolation - Cell Biology and Cell Culture

mouse hepatocytes isolation - Cell Biology Forum. Cell Culture Forum. Post and ask questions about cell culturing, cell lysis, cell transfection, cell growth, and cell biology.


Reply
 
LinkBack Thread Tools Display Modes
  #21  
Old 07-08-2009, 05:05 PM
DBI DBI is offline
Pipette Filler
Points: 76, Level: 1 Points: 76, Level: 1 Points: 76, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2009
Posts: 6
Thanks: 2
Thanked 0 Times in 0 Posts
Default Re: mouse hepatocytes isolation

Hi,
did you end up doing it through the heart? It has been 3 years since then i guess but i am having the same issue..

DBI
Reply With Quote
  #22  
Old 07-08-2009, 05:06 PM
DBI DBI is offline
Pipette Filler
Points: 76, Level: 1 Points: 76, Level: 1 Points: 76, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2009
Posts: 6
Thanks: 2
Thanked 0 Times in 0 Posts
Default Re: mouse hepatocytes isolation

Hi,
did you end up doing it through the heart? It has been 3 years since then i guess but i am having the same issue..

DBI
Reply With Quote
  #23  
Old 07-09-2009, 05:48 PM
Pipette Filler
Points: 1,576, Level: 23 Points: 1,576, Level: 23 Points: 1,576, Level: 23
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2008
Posts: 14
Thanks: 0
Thanked 4 Times in 4 Posts
Default Re: mouse hepatocytes isolation

Hi
I had very low success rate with heart perfusion. I now do all my perfusions via portal vein. (as described in post #20)
Nick
Reply With Quote
The Following User Says Thank You to NickW For This Useful Post:
DBI (07-14-2009)
  #24  
Old 07-14-2009, 11:24 PM
DBI DBI is offline
Pipette Filler
Points: 76, Level: 1 Points: 76, Level: 1 Points: 76, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2009
Posts: 6
Thanks: 2
Thanked 0 Times in 0 Posts
Default Re: mouse hepatocytes isolation

Thanks Nick! So i didn't have any luck with portal vein so far. So i tried doing it through the vena cava and cutting portal vein method. The kidney burst during the procedure although we had clamped the needle. The color of the liver did eventually change a bit but it was not fluffy or anything like that. At this point i am not even sure if it is the collagenase that is causing the problem(sigma from clostridium..C5138). I think i am still having trouble with the path of the solution. Do you think it makes sense to practice with a saline solution first to get the basics of the needle insertion? Because if the path is correct the liver should get pale even with the saline solution only.. right..?
Reply With Quote
  #25  
Old 07-15-2009, 05:33 AM
Pipette Filler
Points: 1,576, Level: 23 Points: 1,576, Level: 23 Points: 1,576, Level: 23
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2008
Posts: 14
Thanks: 0
Thanked 4 Times in 4 Posts
Default Re: mouse hepatocytes isolation

DBI
Yes, practice perfusion through the portal vein; this works the best. You will miss the vein and/or poke all the way through the vein several times before you get it right but after several attepmts you wil succeed. It took me many mice before I got good at it.

Use a new, sharp needle for each mouse for best results.

As soon as you insert into the portal vein, turn on the pump, and snip the vena cava, the liver will turn pale.

I first perfuse with a anti-coagulant perfusion buffer to clear the blood, and then perfuse with collagenase. Saline solution will work for practice, but it is not an anti -coagulant.
Reply With Quote
The Following User Says Thank You to NickW For This Useful Post:
DBI (07-16-2009)
  #26  
Old 07-16-2009, 04:03 PM
DBI DBI is offline
Pipette Filler
Points: 76, Level: 1 Points: 76, Level: 1 Points: 76, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2009
Posts: 6
Thanks: 2
Thanked 0 Times in 0 Posts
Default Re: mouse hepatocytes isolation

I will practice it with saline i guess...

And, previously you had mentioned that you never got ~95% viability. Were you able to increase your viability since then?
Reply With Quote
  #27  
Old 07-17-2009, 07:14 PM
Pipette Filler
Points: 1,576, Level: 23 Points: 1,576, Level: 23 Points: 1,576, Level: 23
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Nov 2008
Posts: 14
Thanks: 0
Thanked 4 Times in 4 Posts
Default Re: mouse hepatocytes isolation

No, I've never gotten better. I still get on average 50-75 % viability.
Let me know if you're able to better results, I would appreciate it.

The portal vein is the best option because it goes directly to the liver; everything pumped in passes through the liver first.
Reply With Quote
  #28  
Old 07-20-2009, 01:13 PM
DBI DBI is offline
Pipette Filler
Points: 76, Level: 1 Points: 76, Level: 1 Points: 76, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2009
Posts: 6
Thanks: 2
Thanked 0 Times in 0 Posts
Default Re: mouse hepatocytes isolation

I will definitely let you know Nick. I found a liver surgeon through my advisor so hopefully i will nail the technical part soon and move on to other issues
Reply With Quote
  #29  
Old 08-05-2009, 08:52 PM
DBI DBI is offline
Pipette Filler
Points: 76, Level: 1 Points: 76, Level: 1 Points: 76, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Jul 2009
Posts: 6
Thanks: 2
Thanked 0 Times in 0 Posts
Default Re: mouse hepatocytes isolation

Well the surgeon still didn't get back. I think he is out there busy with making money...
I am still stuck in the technical part. Everytime i insert the butterfly needle it looks like the major of the solution goes into other body parts like stomach or kidney. I couldn't locate the splenic vein or bile duct to ligate and the technician could locate it either. Any suggestions on this issue?
Reply With Quote
  #30  
Old 11-03-2010, 09:21 PM
Pipette Filler
Points: 1,518, Level: 23 Points: 1,518, Level: 23 Points: 1,518, Level: 23
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Oct 2008
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: mouse hepatocytes isolation

Hi, I'm doing mouse liver perfusions and I had a question about your protocol posted --- why don't you recommend cutting through the middle of the rib cage? I've done many successful perfusions catheterizing through the left ventricle of the heart -- it's very easy to find and once it's in, I tape down the catheter/tubing and my hands are free to use. I cut the portal vein for out-flow. Also, you recommend starting perfusion 5-10 min after death -- you really should start dissection while the animal is down with isofluorane (deeply) and the heart should still barely be beating. Then you cut through the diaphram just before cannulating the heart.
Reply With Quote
Reply

Tags
hepatocytes , isolation , mouse


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
can I use mouse antibody to do immunohistochemistry in mouse brain sections? llzz Antibody Forum 1 03-12-2013 09:02 PM
RNA isolation mouse brain punches Maz RNA Techniques Forum 1 03-10-2010 07:36 PM
Isolating fetal mouse hepatocytes = possible? Lauren Cell Biology and Cell Culture 0 05-26-2009 08:28 PM
DNA isolation from mouse tails and PCR genotyping ivanjdo DNA Forum 0 02-08-2009 01:17 AM
FACS Isolation of Macrophages from Mouse proatpcr Flow Cytometry Forum 1 06-23-2008 05:44 AM


All times are GMT. The time now is 03:19 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.16822 seconds with 16 queries