A method for the recognition and separation of human blood monocytes on density gradients

Abstract

The density distribution of human mononuclear blood leukocytes was studied in order to define the optimal conditions for the separation of monocytes and lymphocytes by isopycnic centrifugation. Under standardized conditions, two populations of cells with partially overlapping, normally distributed densities were consistently found. The cells with the lowest density were recognized as monocytes, using phagocytosis and size distribution analysis as criteria. Since the density of monocytes continuously increased during the centrifugation, optimal separation of monocytes and lymphocytes could only be achieved by limiting the time of centrifugation to 10 min at 2200 g and 4 degrees C. The separation on discontinuous density gradients decreased when the load exceeded 8 X 10(6) mononuclear cells per sq cm. Analysis of the composition of the two cell populations obtained after separation on a three-layer discontinuous gradient revealed that the contamination of the monocytes with lymphocytes was due to the partial overlapping density distributions of both cell types. A small and a large scale method for isolation of monocytes from blood on discontinuous density gradients are presented. Under the described conditions, a preparation of functionally intact monocytes can be obtained which is comparable, both in yield and purity, to those obtained by methods based on surface adherence without the drawbacks of the latter methods.

Separation of Leucocytes: Improved Cell Purity by Fine Adjustments of Gradient Medium Density and Osmolality

“…Different methods for isolation of monocytes have been sutnmarized elsewhere [I2J. Density gradient techniques are based upon the observation that monocytes have a slightly lower density than lymphocytes [9]. However, because the densities of the two types of cells overlap, it is diffieult to establish a satisfactory separation with good yield based on density differences alone.…”

“…I) mononuclear cells (lymphocytes and monocytes = MNC) have a lower density than granulocytes and efficient separation is achieved by using a separation fluid of intermediate density. Mostly iodinated X-ray media [2][3][4][5][6][7][8][9] or polyvinylpyrrolidone-coated silica gel particles [10,11] with a density of about 1.077 g/ml are used as gradient material. Due to the lower density of monocytes than lymphocytes it is possible to separate the two by density gradient separation.…”

“…In practice, however, monocyte purity and yield vary considerably [ 12]. probably due lo overlapping deiisiiies of these two cell populations [9], as well as individual variations of cell density. Such problems also hamper isolation of other subsets of white cells (lymphocytes, basophils.…”

Characterization and Isolation of Very Small Embryonic-Like (Vsel) Stem Cells Obtained From Human Peripheral Blood

“…Son yıllarda insan embriyonik kök hücrelerinin pluripotansiyel karakterine sahip ve organizmada tüm yaşam boyunca var olan yeni bir kök hücre türü tanımlanmıştır (3). Çok küçük embriyonik benzeri kök hücreler (Very small embryonic-like stem cells -VSEL) olarak adlandırılan bu hücrelerin, yetişkin bireyde şimdilik kemik iliği, kordon kanı ve periferik kanda bulunduğu gösterilmiştir (4). Bu hücrelerin izolasyon işlemi esnasında mononükleer tabakanın dışında eritrosit katmanında bulunduğu için kaybedildiği iddia edilmektedir.…”

The Use of Frozen Monocytes in Phagocytosis Studies

“…Monocytes have a lower average density than other cells in human blood (13), and many methods of monocyte isolation based on density differences have been developed (1, 2,4, 5, 8, 1 1,12,24,25). However, owing to overlapping predominantly by lymphocytes, efficient separation based on density gradient centrifugation alone cannot be acheived.…”