Non-isopycnic cell purification using percoll

Abstract

A method of “non-isopycnic” cell isolation and purification has the steps of adding a sample of blood to a defined volume to a corresponding ratio volume of EDTA solution to produce a volume of anti-coagulated blood; taking a predetermined volume of the anti-coagulated blood and placing it in a first tube containing a selected defined volume of PSS, wherein the selected defined volume of PSS is taken from a group of defined volumes of PSS, as each defined volume of PSS establishes a specific cell type to be purified; centrifuging the tube for a first predetermined time and speed to form a volume of supernatant of plasma/PSS and a bottom sedimented volume of mostly red blood cells; extracting the supernatant to within a proximity of an interface between the sedimented red blood cells and the supernatant; transferring an appropriate, pre-selected volume amount of the supernatant into a second tube holding a defined volume of physiological media; mixing the solutions gently; centrifuging for a second predetermined speed and time; and pouring off the supernatant wherein at the bottom of the tube will be a cell button containing a volume of the selected purified cells in a high percentage and a very small quantity or low percentage of some contaminating non-selected cells. This method is useful in establishing high purity concentrations of selected cells from whole blood such as monocytes, lymphocytes, neutrophils and basophils.

1 . One or more kits of prepared tubes for non-isopycnic cell purification, comprising:
 one or more kits selected from a group of kits including a kit for monocytes, a kit for lymphocytes, a kit for neutrophils and a kit for basophils, wherein each kit includes a selected defined volume of PSS contained in each of one or more first tubes and a selected defined volume of physiological media containing RPMI 1640 media, 25 mM HEPES, Human Serum Albumin and ethylenediaminetetraacetic acid or EDTA, collectively referred to as RHAE in each of one or more second tubes.   
   
   
       2 . The one or more kits of prepared tubes for non-isopycnic cell purification of  claim 1  wherein the kit for basophils further comprises:
 one or more third tube containing a selected defined volume of PSS, each third tube being a 5 ml round bottom plastic tube.   
   
   
       3 . The one or more kits of prepared tubes for non-isopycnic cell purification of  claim 1  wherein each of the first or second tubes is a 50 ml conical bottom plastic tube. 
   
   
       4 . The one or more kits of prepared tubes for non-isopycnic cell purification of  claim 3  wherein the kit for monocytes cells has 5 ml of PSS in each first tube and 5 ml of RHAE in each second tube. 
   
   
       5 . The one or more kits of prepared tubes for non-isopycnic cell purification of  claim 3  wherein the kit for neutrophils cells has 20 ml of PSS in each first tube and 6 ml of RHAE in each second tube. 
   
   
       6 . The one or more kits of prepared tubes for non-isopycnic cell purification of  claim 3  wherein the kit for basophils cells has 10.25 ml of PSS in each first tube and 0.6 ml of RHAE in each second tube. 
   
   
       7 . The one or more kits of prepared tubes for non-isopycnic cell purification of  claim 2  wherein the kit for basophils cells has 0.575 ml of PSS in each third tube.