I had to isolate lymphocytes from human blood for further cytofluorimetric analysis. I used the Lympholyte-mammal protocol but from 15cc of human blood I had a low yield recovery of lymphocytes (3-4*106, my coworkers tell me that they usually recovered 10-12*106).

I diluted 1:1 15cc of blood with RPMI, I put 14-15 ml of Lympholyte in two 50 ml centrifuge tubes, then I carefully layered the diluted blood with a pipette over the Lympholyte.

Centrifuge at 2500 rpm for 20 min at RT.

Remove the lymphocyte layer at the interface and transfer to another tube, wash with RPMI at 2500 rpm for 10min at RT.

Count the cells.

I used the Lympholyte at RT, I centrifuged at 25°C, and I performed isolation from blood at RT, after sample taking.

My questions:

Is RPMI a good choice or is it better to use PBS (w/o Ca and Mg)? My co workers used undiluted blood but I have an even worse recovery.

Is it better to reduced the amount of initial diluted blood (15ml over 14ml of Lympholyte), maybe only 10 ml or less?

When I carefully layered the diluted blood over the Lympholyte (I take some minutes) the blood started to decant, under the Lympholyte, spontaneously (as bloody drop), even if I add only 4-5ml. Why?

In some protocols I have found that the they remove the upper layer that contains the plasma and platelets and then transfer the mononuclear cell layer in another tube. My lymphocyte layer, after the first centrifugation, is not a well-defined ring but it is a sort of white cloud. Is this normal? In this case how can I remove the upper layer without losing the lymphocytes?