Thread View: sci.med.immunology
5 messages
5 total messages
Started by Bryan Heit
Wed, 15 Nov 2000 00:00
Netrophil isolation problem
Author: Bryan Heit
Date: Wed, 15 Nov 2000 00:00
Date: Wed, 15 Nov 2000 00:00
14 lines
1534 bytes
1534 bytes
I'm having a problem isolating neutrophils. I'm purifying neutrophils from human blood using dextran sedimentation, followed by hypotonic lysis, with final isolation using fycol-hypaque centrifugation. My problem is with the dextran sedimentation stage. I'm using 6% dextran (15g dextran + 2.25g NaCl + 250ml ddH2O). What should be happening is a sedimentation lasting approximatly 30min. This should result in a layer of RBC free supernatant with leukocytes suspended in it. what's happening is sedimentation takes over and hour and I get a supernatant with almost no cells in it at all. I've attempted to draw off the supernatant as it forms, but I still end up with no leukocytes in the supernatnat. Does anyone know what's wrong? Bryan
Re: Netrophil isolation problem
Author: Alberto Clivio
Date: Thu, 16 Nov 2000 00:00
Date: Thu, 16 Nov 2000 00:00
13 lines
1356 bytes
1356 bytes
Dear Bryan, I think there are 2 solutions to your problem: 1) check the molecular weight of your Dextran: it usually works very well with high molecular weight dextran (>500.000). 2) you might use a special "Mono-poly resolving medium" you can get from different companies, which avoids the dextran separation step and gives two sharp bands of white cells: the upper band is mononuclear cells, the lower one granulocytes. RBC sediment at the bottom of the tube. Alberto Clivio Milano, Italy
Re: Netrophil isolation problem
Author: Bryan Heit
Date: Thu, 16 Nov 2000 00:00
Date: Thu, 16 Nov 2000 00:00
27 lines
1800 bytes
1800 bytes
Does that method of isolation activate the neutophils? My experiment requires unactivated neutrophils, which is why I chose dextran sedimentation over other methods. Also, can the Mono-poly resolving medium be used with whole (and anticoagulated) blood, or is another purification satep required beforehand? Bryan On Thu, 16 Nov 2000, Alberto Clivio wrote: > Dear Bryan, > > I think there are 2 solutions to your problem: > 1) check the molecular weight of your Dextran: it usually works very well with > high molecular weight dextran (>500.000). > > 2) you might use a special "Mono-poly resolving medium" you can get from > different companies, which avoids the dextran separation step and gives two > sharp bands of white cells: the upper band is mononuclear cells, the lower one > granulocytes. RBC sediment at the bottom of the tube. > > Alberto Clivio > Milano, Italy > > >
Re: Neutrophil isolation problem
Author: Alberto Clivio
Date: Fri, 17 Nov 2000 00:00
Date: Fri, 17 Nov 2000 00:00
9 lines
1300 bytes
1300 bytes
Dear Bryan, the Mono-Poly resolving medium does not activate neutrophils, it is just a special solution of Ficoll-Paque with a higer density as compared to the usual one. It must be used with whole blood (both heparinized and with Ca2+ chelating agents) and it works as a single step purification procedure. Good luck Alberto Clivio
Re: Netrophil isolation problem
Author: Alberto Clivio
Date: Fri, 17 Nov 2000 00:00
Date: Fri, 17 Nov 2000 00:00
11 lines
1341 bytes
1341 bytes
Dear Bryan, the Mono-Poly resolving medium does not activate neutrophils, it is just a special solution of Ficoll-Paque with a higer density as compared to the usual one. It must be used with whole blood (both heparinized and with Ca2+ chelating agents) and it works as a single step purification procedure. Good luck Alberto Clivio
Thread Navigation
This is a paginated view of messages in the thread with full content displayed inline.
Messages are displayed in chronological order, with the original post highlighted in green.
Use pagination controls to navigate through all messages in large threads.
Back to All Threads