Dextran sedimentation

Figure 7.7. Expression of CR3 in neutrophils in unfractionated blood and after isolation. The expression of CD1 lb was measured in neutrophils in (i) unfractionated whole blood, (ii) after isolation using Mono-Poly resolving medium and (iii) after isolation using dextran sedimentation and centrifugation in Ficoll-Hypaque. Further experimental details may be found in Watson, Robinson Edwards, 1992.

Isolation of human neutrophils. Leukocytes were obtained from normal donors by leukapheresis with an IBM 2997 Blood Cell Separator (8). Normal mature neutrophils were purified from this mixed leukocyte preparation by dextran sedimentation and Ficoll-Hypaque gradient centrifugation as previously described (9). The Wright-stained smears of the preparation showed that the cells were over 95% neutrophils. 

During the dextran sedimentation, prepare the following in 50-mL conical tubes (1) 35 mL of 0.09% NaCl, (2) 20 mL of 1.7% NaCl, (3) 12 mL of Ficoll-Paque , and (4) 20 mL of injection- or irrigation-grade water. These solutions are normally stored at 4 °C, but to prevent priming and aggregation of PMNs, it is optimal to warm the solutions to room temperature. 

Mixed leukocytes are prepared using dextran sedimentation of erythrocytes, centrifugation of leukocytes from the plasma, and lysis of residual erythrocytes with ammonium chloride. Granulocytes and mononuclear leukocytes are separated from each other and from erythrocytes by density gradient centrifugation using a mixture of Ficoll, Hypaque and dextran. Thirty ml of blood provides sufficient leukocytes for study of adenine, guanine and hypoxanthine metabolism in duplicate. Suspension cultures of human lymphoblasts and leukemic cells, and monolayer cultures of skin fibroblasts and amnionic cells were studied under normal culture conditions approximately 10 cells are required for each incubation. 

Evidence that this is the case has also been obtained from sedimentation velocity studies on dextrans where the sedimentation coefficient S, which is proportional to M/f21, is shown to be molecular weight-independent at high concentrations 15). Furthermore, studies on the intradiffusion of tritiated water (HTO) in dextran solution8) (footnote 2) revealed that the term... 

Leukocytes are prepared from EDTA-blood (approximately 3-5 ml) by the density gradient method. EDTA-blood is mixed by inversion and 5 ml is slowly added to 1 ml dextran solution. The blood and dextran solution are carefully mixed so that formation of foam is avoided. The mixture is allowed to stand for 1 h. If further time for sedimentation is required, it has to be noted as it may affect the resulting enzymatic activities. The time needed for proper sedimentation depends on sample quality and should not exceed 3 h. The upper phase including the white cells is transferred to another tube and spun at 600-1000 g for 10 min. The supernatant is... 

Figure 1. Effects of the concentration of dextrans with various molecular weights on three indices of BBC aggregation (16). MAI indicates the average number of RBCs in each aggregation unit counted under the microscope. ESR is the maximum rate of sedimentation of erythrocytes in a calibrated tube, with corrections made for changes in viscosity and density of the suspending medium following the addition of dextrans. The relative viscosity (t)r) is the ratio of the viscosity of RBC suspension to that of the suspending medium at a shear rate fo 0.1 sec 1. The RBC concentration of the suspension was 1% for MAI and 45% for ESR and r)r measurements. The vertical bars represent SEM. (A), Dx 40 (O), Dx 80 (M), Dx 150 (A), Dx 500 ( ), Dx 2000.

A considerably smaller value for the molecular weight of the methylated product of this dextran has been obtained by Hassid and Barker.64 From viscosity measurements (using Km = (10)-3 for methyl cellulose),66 the molecular weight was roughly indicated as 11,700, whilst the value from sedimentation equilibrium measurements was 3,275 (although the latter figure was not considered very reliable). 

Isolation of neutrophils polymorphonuclear (PMN) can be done using citrate/ dextran/gelatine sedimentation (see Note 1). The following compositions are used ... 

The molecular-sieving effect on proteins has been very clearly demonstrated by Pedersen (1962). Bovine serum albumin, obtained by fractionated ammonium sulfate precipitation, was filtered through a column of cross-linked dextran of water regain 15 gm/gm (Sephadex G-200). Monomer and dimer forms were easily separated from each other and from other components of larger sedimentation constants (Fig. 8). Heterogeneity in the monomer as well as in the dimer preparation was indicated... 

Enzyme source (see Note 4) Chinese hamster ovary cells (CHO-Kl, American Type Culture Collection) were transiently transfected via standard DEAE-dextran methods with an expression construct encoding the human ECE-lc protein. The medium was changed to serum-free medium the day after transfection. Two days after transfection, cells were harvested and a crude membrane fraction sedimented by ultracentrifugation (100,000g for 1 h). The pellet was suspended in assay buffer, aliquoted, and frozen at -70°C until assay. Protein concentration was determined by standard methods (e.g., Lowry or BCA). The equivalent of 3 pg total protein was used in each assay well. 

Obtain a fresh blood sample anticoagulated with ACD-A. Maintain the blood sample at room temperature do not refrigerate. The cell isolation should be performed as soon as possible after the sample is collected. Add 3 mL of 5% dextran/10 mL of blood, gently mix, and let stand at room temperature for 40-45 mm to allow red blood cell sedimentation. After the sedimentation step, carefully pipet off the straw-colored plasma layer to within =0.5 cm of the red blood cell interface. 

Osmotic nephrosis Mannitol, immune globulin, dextrans, hetastarch Urine sediment shows vacuole containing cells... 

The prediction has been made that increases in the fractional volume occupancy of macromolecules in a physiological fluid should non-specifically accelerate the formation of amyloid by any amyloidogenic protein. Hatters et al. (2002) have looked at the effect of dextran TIO solutions on amyloid formation by human apolipoprotein C-II. The dextran was shown by analytical sedimentation equilibria studies not to form heterocomplexes with the apoC-n and nor was it incorporated into any of the amyloid fibrils studied. 

---