4 replies to this topic

[berrycom]

It's nice meeting all of you here. This is my first post here.

I have a question concerning hematopoietic stem/progenitor cells enrichment kits. Has anybody tried to do lineage depletion in spleen or bone marrow to enrich Hematopoietic stem/progenitor cells. If yes, which kits have you used? I know there are many kits out there but I will be grateful to hear from someone who had experience.

Many thanks in advance,
berricom

Edited by berrycom, 08 October 2009 - 07:17 PM.

[TracyDuke]

Try https://www.miltenyi...en/default.aspx. Click on the first box that says" cell separation" on the upper left corner on the mainpage. They have positive and negative selection reagents and kits, depending on your needs. A lot of literatures use their products.

[berrycom]

TracyDuke, on Oct 19 2009, 01:54 PM, said:

Try https://www.miltenyi...en/default.aspx. Click on the first box that says" cell separation" on the upper left corner on the mainpage. They have positive and negative selection reagents and kits, depending on your needs. A lot of literatures use their products.

Hi TracyDuke,
Many thanks for your reply. Actually, I have tried Miltenyi lineage depletion kit but I had a viability issue. After one day of culture approximately 60% cells were dead (trypan blue exclusion). Has anybody used this kit and cultured the hematopoietic progenitors afterwards? I had many samples so the selection process was quite lengthy. Maybe that was the problem. I need to repeat it.

Best regards,
berrycom

[boudou_611]

berrycom, on Nov 17 2009, 05:15 AM, said:

TracyDuke, on Oct 19 2009, 01:54 PM, said:

Try https://www.miltenyi...en/default.aspx. Click on the first box that says" cell separation" on the upper left corner on the mainpage. They have positive and negative selection reagents and kits, depending on your needs. A lot of literatures use their products.

Hi TracyDuke,
Many thanks for your reply. Actually, I have tried Miltenyi lineage depletion kit but I had a viability issue. After one day of culture approximately 60% cells were dead (trypan blue exclusion). Has anybody used this kit and cultured the hematopoietic progenitors afterwards? I had many samples so the selection process was quite lengthy. Maybe that was the problem. I need to repeat it.

Best regards,
berrycom

Hi Berrycom,
I have some experience with miltenyi lineage depletion kit, to be specific: CD133 microbead isolation kit. I also have some issue with the viability and purity. To improve the viability, I have increase the BSA to 5-10 % in the buffer plus make sure that the buffer is cold during the isolation procedure been carried out. And to obtain the purer population, I run down the cells suspension thru the isolation column twice. So far it is work.

But recently I talked to one competitor who is selling the isolation product also. he said isolation kit from miltenyi using dextran particles/beads to bind to cells that might interfere when you are about to identify/characterize the isolated cells using flow cytometry. whereas theirs, using nano particles/beads that wont interfere in fact, flow cytometry couldnt able to identify/ pick up the particles. i do not know how true it is..i havent really find out about it yet..

Rgrd,
boudou

[signstem]

boudou_611, on Nov 20 2009, 01:15 PM, said:

berrycom, on Nov 17 2009, 05:15 AM, said:

TracyDuke, on Oct 19 2009, 01:54 PM, said:

Try https://www.miltenyi...en/default.aspx. Click on the first box that says" cell separation" on the upper left corner on the mainpage. They have positive and negative selection reagents and kits, depending on your needs. A lot of literatures use their products.

Hi TracyDuke,
Many thanks for your reply. Actually, I have tried Miltenyi lineage depletion kit but I had a viability issue. After one day of culture approximately 60% cells were dead (trypan blue exclusion). Has anybody used this kit and cultured the hematopoietic progenitors afterwards? I had many samples so the selection process was quite lengthy. Maybe that was the problem. I need to repeat it.

Best regards,
berrycom

Hi Berrycom,
I have some experience with miltenyi lineage depletion kit, to be specific: CD133 microbead isolation kit. I also have some issue with the viability and purity. To improve the viability, I have increase the BSA to 5-10 % in the buffer plus make sure that the buffer is cold during the isolation procedure been carried out. And to obtain the purer population, I run down the cells suspension thru the isolation column twice. So far it is work.

But recently I talked to one competitor who is selling the isolation product also. he said isolation kit from miltenyi using dextran particles/beads to bind to cells that might interfere when you are about to identify/characterize the isolated cells using flow cytometry. whereas theirs, using nano particles/beads that wont interfere in fact, flow cytometry couldnt able to identify/ pick up the particles. i do not know how true it is..i havent really find out about it yet..

Rgrd,
boudou

we used the miltenyi kit before (the one for purifying progenitor) and more than 99% of cells can be depleted; we improve the viability by using the right medium, instead of BSA as buffer.