Red Cells in Clear Ficoll Layer Following Density Centrifugation - Monocyte Isolation (Sep/18/2007 )

Hi All,

I am experiencing problems with red blood cell contamination following density gradient centrifugation of buffy coats. There are supposed to be 4 layers that form when the buffy coat concentrate is centrifuged on histopaque/ Ficoll - the plasma layer, the white cell layer, the clear ficoll/ histopaque layer, and the red blood cell layer. My histopaque/ficoll layer is not clear. It has alot of red blood cells in it. Even though I am carefully removing the white cell layer I am still getting alot of red cells in my white cell pellet because of the contaminated ficoll/histopaque layer. I have tried ammonium chloride lysis to remove the red cells but there are still too many red cells. There are so many that i cannot do cell counts of mononuclear cells. Does anyone know what I am doing wrong? Does anyone have advice?

Thanks.

Some points to remember

- if the height of the blood sample in the tube is too much it can give red cell contamination. I don't know the right ratio but we use 15ml Falcon with 5ml Ficoll and 10ml blood sample.

- temperature of Ficoll. Around 20 deg will give optimal result. If it is too warm, you will lose white cells.

- dilute the sample appropriately. ~ 2-4 times with PBS

I thinck you have to be sure that the blood is diluted 1:1 with PBS
Be sure bout the temperature 20-22degree
finally be sure about the centrifugation speed 3200RPM and the time of centrifugation 30min