Separation of Human Lymphocytes from Citrated Blood by Density Gradient (NycoPrep) Centrifugation: Monocyte Depletion Depending upon Activation of Membrane Potassium Channels

Abstract

Routine one-step centrifugation procedures (Lymphoprep LP, Percoll) commonly used for separation of blood cells split the cells into two major fractions. After centrifugation the mononuclear cells (MNC monocytes and lymphocytes) are located on the top of the separation fluid, whereas erythrocytes and granulocytes have sedimented to the bottom. We now show that a relatively pure lymphocyte suspension can be obtained by one-step centrifugation of citrated blood by using NycoPrep (NP iohexol), a nonionic X-ray contrast agent. With this gradient medium also the monocytes pass to the bottom, leaving lymphocytes on the top. In parallel separations with LP, which contains Ficoll and a fully dissociated sodium salt of a contrast medium, the results were as usual, i.e. $70±85% lymphocytes and 30±15% monocytes in the top fraction. The monocyte depletion with NP depended upon the use of citrated (ACD) blood and a proper balance of density and osmolality of the gradient medium, and was enhanced by 20 min preincubation with CaCl 2 at room temperature. Monocyte depletion could not be obtained with LP. Under optimal conditions (density 1.075 g/ml, osmolality 280±300 mOsm/kg), the monocyte admixture amounted to $1 (0±2)%, in separations with buffy coat samples. For freshly drawn blood, it was necessary to slightly modify the NP solution. The monocyte depletion was counteracted by blockers of K channels or by KCl in the cell suspension. Following incubation in NP of Percoll-separated cells, an enhanced release of K was observed. The results are interpreted as follows: NP mediates the opening of K channels of MNC, which leads to efflux of K , accompanied with associated anions (Cl ± ). This reduces the osmolality inside the cells which therefore expel water to maintain osmotic equilibrium. In this regard it appears that monocytes are more sensitive than lymphocytes, their density therefore increasing more, so that they are able to pass the density barrier otherwise exerted by the gradient medium.

Noninvasive management of the diabetic foot with critical limb ischemia: current options and future perspectives

“…In the majority of studies, bone marrow is the primary source of stem cell material and enrichment of mononuclear cells from the crude aspirate can be accomplished with different techniques such as density gradient centrifugation (Ficoll™) [Boyum et al 2002] or other commercially available blood-centrifugation and plasmapheresis systems [Tateishi-Yuyama et al 2002]. However, these are laborious and require the background of a specialized and certified hematological or immunological unit.…”

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

“…Nycoprep™ and OptiPrep™ are gradient solutions without Ficoll™, based on a tri‐iodinated derivative of benzoic acid with three aliphatic, highly hydrophilic side chains or on iodixanol, respectively. They thus are not based on a polysaccharide net . From the granulocyte/erythrocyte mix, neutrophil granulocytes can be isolated further by dextran sedimentation , and erythrocytes lysed by hypotonic shock (see Chapter IV, section 2.5).…”

Guidelines for the use of flow cytometry and cell sorting in immunological studies*

“…Nycoprep™ and OptiPrep™ are gradient solutions without Ficoll™, based on a tri‐iodinated derivative of benzoic acid with three aliphatic, highly hydrophilic side chains or on iodixanol, respectively. They thus are not based on a polysaccharide net . From the granulocyte/erythrocyte mix, neutrophil granulocytes can be isolated further by dextran sedimentation , and erythrocytes lysed by hypotonic shock (see Section II.1.5).…”