Anticoagulated whole

Obtain a fresh blood sample anticoagulated with ACD-A. Maintain the blood sample at room temperature do not refrigerate the blood prior to study. The assay should be performed within 5 h after the blood is collected. Pipet 100 pL of anticoagulated whole blood into sterile, 5.5-mL polypropylene tubes immediately before initiating the phagocytosis assay. 

Add 200 oL of anticoagulated whole blood to a 5.5-mL polypropylene sterile tube. 

Incubate 1 mL of anticoagulated whole blood with 2, 7 DCFH-DA (final concentration of 50 xM) for 10 min at 37°C in a shaking water bath. 

Isolation of Human Platelets The steps described in the subsequent subheading outline the procedure for isolation and purification of platelets from whole blood obtained by venipuncture from human volunteers. Obtain blood sample by venipuncture from an antecubital vein into polypropylene syringes containing either sodium citrate (0.38 % final concentration) or heparin (10 U/ml final concentration). Centrifuge anticoagulated whole blood at 160 x g for 15 min to prepare platelet-rich plasma (PRP). 

The cone-and-plate viscometer is an in vitro flow model used to investigate the effects of bulk fluid shear stress on suspended cells. Anticoagulated whole blood specimens (or isolated cell suspensions) are placed between the two platens (both of stainless steel) of the viscometer. Rotation of the upper conical platen causes a well-defined and uniform shearing stress to be applied to the entire fluid medium as described by Konstantopolous et al. (1998). The shear rate (y) in this system can be readily calculated from the cone angle and the speed of the cone using the formula i/ = where y is the shear rate in sec-1, mis the... 

Incubate anticoagulated whole blood with platelet antagonist or vehicle (control) at 37 °C for 10 min. 

Add the fluorescent dye quinacrine dihydrochloride to anticoagulated whole blood samples at a final concentration of 10 pM immediately after blood collection. 

Perfuse anticoagulated whole blood through the flow chamber for 1 min at wall shear rates ranging... 

A variety of assays have been developed to quantify phagocytic activity. These include direct microscopic visualization (2,3), spectropho-tometric evaluation of phagocytized paraffin droplets containing dye (4), scintillation counting of radiolabeled bacteria (5), fluorometric (6), and flow cytometric analysis of fluorescent particles (7-13). The flow cytometric assay offers the advantage of rapid analysis of thousands of cells and quantification of the internalized particle density for each analyzed cell. The assay may be performed with purified leukocyte preparations (7-13) or anticoagulated whole blood (14,15). 

Pipet 100 pL of anticoagulated whole blood into sterile, 5.5-mL polypropylene tubes immediately before initiating the phagocytosis assay. 

Sample material EDTA, heparin or citrate anticoagulated whole blood. 

Add 10 mL of EDTA-anticoagulated whole blood to a 15 mL centrifuge tube and spin the tube with 1500g at 4°C for 30 min (see Note 11). 

Eggesbo JB, Hjermann I, Hostmark AT, Kierulf P. LPS induced release of IL-1 P, IL-6, IL-8 and TNF-a in EDTA or heparin anticoagulated whole blood from persons with high or low levels of serum HDL. Cytokine 1996 8 152-60. 

Table IV. Platelet Accumulation from Heparin Anticoagulated Whole Blood on Glass Capillary Surfaces Precoated v/ith Purified Human Albumin, Hnman Fibrinogen or Bovine Collagen. Quantitaticr of Platelet Deposition by Badioimmuroassay with Iodine Labeled Monoclonal Antibody 6C9 against Human Platelet Glycoprotein Complex Ilb-IIIa...

In summary, the capillary perfusion system represents a simple in vitrc technique for the quantitation of protein adsorption and platelet accumulation on artificial surfaces under well defined hydrodynamic conditions (0-4.,000 s ). Radioisotopic methods enable simultaneous study of platelet deposition and adsorption of one or more plasma proteins. Experiments may be performed using washed platelet suspensions or anticoagulated whole blood, platelet deposition being measured in the latter case by surface phase radioimmunoassay with a... 

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