Human monocytes were isolated from the cellular residues remaining after plateletapheresis of donors using an automated blood cell processor. Mononuclear cells were obtained with density gradients ...and separated from lymphocytes by stepwise elutriation. The isolated cells were frozen using extracellular hydroxyethyl starch and intracellular dimethylsulfoxide. In three procedures, approximately 1 X 10(9) monocytes were obtained. Ninety-nine percent of isolated monocytes were viable in a fluorescein diacetate (FDA)-ethidium bromide (EB) test. Myeloperoxidase-positive cells were 95 percent and 90 percent in the two chambers. Ninety-four percent of monocytes ingested five or more opsonized polycyclic hydrocarbon particles and 95 percent ingested one or more ethidium-treated zymosan particles. After storage in liquid nitrogen for up to 9 weeks, 99 percent of the cells were recovered after thawing. Of these, 95 percent were myeloperoxidase-positive, 94 percent showed intact membranes in the FDA-EB test, 95 percent ingested five or more opsonized polycyclic hydrocarbon particles, and 96 percent ingested one or more ethidium-treated zymosan particles. These results demonstrate the utility of elutriation as a means to isolate large numbers of monocytes. The isolated cells can be cryogenically preserved for at least 2 months with small loss of phagocytic function.
Crystals of the mouse major urinary protein (MUP) and rat alpha-2u globulin (AMG) have been grown from solutions of polyethylene glycol 3350 and CdCl2, respectively. The crystals differ both in their ...morphologies and space groups but have very similar unit cell sizes. AMG crystallized in P2(1) (a = 56.6 A, b = 103.8 A, c = 62.7 A, beta = 95.1 degrees) with four subunits/asymmetric unit, while MUP gave crystals in P4(1)2(1)2 or P4(3)2(1)2 (a = 57.3 A, c = 109.9 A) with one subunit/asymmetric unit. Both crystal forms diffract beyond 2.8 A resolution.
Fluorescent particles (Fluolite) with an average size of 0.1 micrometers were ingested by human granulocytes after incubation in fresh normal human serum (NHS). Ingestion was assessed by visual ...counting in a fluorescent microscope of cells containing particles. Ingestion required fresh normal serum and did not occur when serum was heated for 30 min at 50 degrees C or in the presence of ethylenediaminetetraacetic acid (EDTA). It did not occur in serum genetically deficient in C3b inactivator or in C3. Phagocytic activity was restored to C3-deficient serum by purified human C3 and to heat inactivated serum by purified factor B. Opsonic activity was present in NHS containing 5 mM Mg++ and 10 mM ethyleneglycoltetraacetic acid (EGTA) and in human serum genetically deficient in human C components C2 and C5. Agammaglobulinemic sera had normal opsonic activity. Opsonization of particles in this system is mediated through the alternative pathway of C activation, and its measurement serves as a simple quantitative functional assay for this system.
Scanning microscopic and functional studies were made of granulocytes isolated from CPD anticoagulated whole blood by counterflow centrifugation in a Beckman JE-6 rotor. The collection buffer was ...phosphate (20 mM) buffered saline (280 mOsM) with glucose (29 mM) and human serum albumin (1.2% w/v). The final suspension contained less than 2% mononuclear cells and 5% red cells. Incubation and fixation at various temperatures revealed two distinct temperature dependent shape transformations. At 22, 37, 40 and 45 degrees C granulocytes were ameboid with extensive highly textured veils. These smoothed progressively, bullae and blebs formed, and membranes peeled finally leaving nonfunctional spheres with smooth surfaces. At 4 degrees C, granulocytes were irregular spheres, less rugose but with numerous microvilli and nodules. Veiling was absent. Phagocytosis, initially low, progressively declined over 48 h while cell surfaces become smooth. Some formed blebs, but all terminated as nonfunctional spheres with untextured surfaces containing occasional large single holes. Cellular stability estimated from changes in volume distributions, and phagocytosis by microfluorescence measurements of yeast and latex particle ingestion were also temperature dependent and paralleled the shape progressions. It is concluded that at body (37 degrees C) or fever (40 degrees C) temperatures, granulocytes have dynamic membrane surfaces characterized by extensive veiling and high function. At 4 degrees C they are relatively inactive spheres devoid of pseudopodia or veils, yet functional at slow rates.
Human granulocytes were isolated from blood either by counterflow centrifugation in a Beckman JE-6 rotor or by sedimentation of the red blood cells with dextran and centrifugation of the ...granulocyte-rich plasma. The stability of the granulocytes was assessed during storage in the liquid state by measurements of granulocyte loss, volume distribution characteristics, and ability to produce fluorescein in their cytoplasm and to exclude ethidium bromide from their nuclei. After storage at 4 C for 2 days in phosphate-buffered saline with a pH of 7.1 containing 5 g/dl human albumin + 0.46 g/dl dextrose or 1 g/dl Physiogel + 0.46 g/dl dextrose, the granulocytes were adequately preserved from the in vitro measurements.
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