I did centrifuging of 5 ml of peripheral blood on 5 ml of ficoll 1800 rpm for 35 minutes at 20 ° C. My aim on this experiment is to obtain the whole leukocyte fraction for further lab work. My question is should I get after centrifuging two leukocyte rings? because I got only one in the tube . What is my mistake? Any suggestions please!
Hi Oula,
After the centrifugation you should be able to get two distinct layers of Leukocytes. The first layer immediately above the RBCs; here you get the heavier WBCs like the Granulocytes (Esinophils and Basophils) . The second layer is immediately below the plasma layer; where you can see the PBMCs.
Please have a look at the attached picture, and let me know which layer you are missing.
The single layer you see is the PBMCs, as stated above. The "granulocyte" layer is not that easy to see becuase it is right above (and most times a LOT more spread out) the RBCs, and there is no way to remove that layer without taking up tons of RBCs with it.
If you want the granulocyte layer, you need to add a second gradient to the mix so you can get the granulocytes away from the RBCs.
Dear if you need to work on the whole leukocytes there is no need for distinction in the centrifuged blood.
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