isolating WBC from splenocytes/spleen - (Aug/09/2005 )

It has been years since I have done this so I am asking the forum for a more modern technique.

I take spleen, grind it and push through a 70 µM filter . I now want to remove the red blood cells, endothelial cells and possibly granulocytes. Our lab has Ficoll-Paque but I do not know what volume of FP to volume of cell suspension to use, nor speed and time for centrifuging.

Most protocols talk about isolating WBC from blood but don't mention splenocytes.

What do you guys suggest?

Are there alternatives to Ficoll-Paque?

Thanks,

AussieUSA.

To get rid of RBC's from splenocytes, I use TAC (tri-ammonium chloride buffer) for 5 minutes at 37C...never have used ficoll for this...

It has been years since I have done this so I am asking the forum for a more modern technique.

I take spleen, grind it    and push through a 70 µM filter  . I now want to remove the red blood cells, endothelial cells and possibly granulocytes. Our lab has Ficoll-Paque but I do not know what volume of FP to volume of cell suspension to use, nor speed and time for centrifuging.

Most protocols talk about isolating WBC from blood but don't mention splenocytes.

What do you guys suggest?

Are there alternatives to Ficoll-Paque?

Thanks,

AussieUSA.

Unfortunatley BWHHMS, red cell lysis does not remove endothelial cells and granulocytes. You need to remove using a separation medium like ficoll.

I am looking for Ficoll / Histopaque etc techniques for splenocytes.

Thanks.

I am looking for Ficoll / Histopaque etc techniques for splenocytes.

Thanks.
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me too!!!

Thank You...