Erythrocytic suspension

Addition of sodium cyanide (5 mmol) and tributyltin (10 pmol) to human erythrocyte suspensions resulted in a synergistic increase in tributyltin-induced hemolysis (Gray et al. 1986). Mechanisms are not clear, but may involve elevated pH of high sodium cyanide concentrations. 

Erythrocyte suspension Sample purged absorption of hydrogen cyanide in sodium hydroxide conversion of thiocyanate to cyanide by potassium permanganate oxidation Spectrophotometry (thiocyanate- cyanide determination) No data 93-97 McMillan and Svoboda 1982... 

Djordjevich L, Kashani A, Miller IF, et al. Measurements of viscosity of synthetic erythrocyte suspensions. Biorheology 1987 24 207. 

Erythrocyte suspensions are frozen at -20°C overnight prior to analysis. Pipette 100 pi of the internal standard working solution in a 4-ml glass vial. Add 50 pi of plasma or erythrocyte suspension. Add 1 ml of 3N methanolic HC1, close the vial with a screw cap and Teflon insert and allow the transmethylation to proceed at 90°C for 4 h. 

Using haemoglobin and epoxybutene, Osterman-Golkar et al. (1991) observed the formation of two diastereomeric pairs of adducts to the N-terminal valine of haemoglobin namely V-(2-hydroxy-3-buten-l-yl) valine and V-( I -hydroxy-3 -buten-2-yl)valine. These findings were corroborated by Richardson et al. (1996), who incubated erythrocyte suspensions obtained from mice, rats and humans with epoxybutene. The second-order rate constant of adduct formation for the sum of both adducts (HOBVal) was determined in vitro at 37°C to be 0.29 x 10- L/g globin/h with erythrocytes isolated from mice (Recio et al., 1992 value corrected by the same authors to the one quoted here, Osterman-Golkar et al., 1993). 

M. M. Fromjmovic, A. Okagawa, and S. G. Mason, Rheo-optical transients in erythrocyte suspensions, Biochem. andBiophys. Research Comm., 62, 17 (1975). 

Add 5 ml of the erythrocyte suspension to 25 ml of hypotonic phosphate buffer in a 50-ml plastic centrifuge tube. Cap the tube and invert several times to mix (do not use a Vortex mixer). 

Procedure Packed erythrocytes, washed twice in Hepes buffer, are resuspended in an equal volume of the same buffer. The erythrocyte suspension (2 ml) is carefully layered over the Percoll-diatrizoate solution (2 ml) in a glass centrifuge tube with an internal diameter of 10 mm. After centrifugation at 400 x g for 20 min at room temperature, erythrocytes with normal density are carefully removed from the surface of the Percoll-diatrizoate cushion using a pasteur pipette. This fraction is referred to as the normal density fraction. Erythrocytes with elevated density, which had pelleted below the Percoll-diatrizoate, can then be removed in a similar manner. This latter fraction is referred to as the dense fraction. Finally, each fraction of erythrocytes is washed twice in 10 vol. of Hepes buffer at 4°C. 

Hemagglutination tests are generally conducted at room temperature by adding a drop of a 2 to 3% erythrocyte suspension to tubes or wells containing serially diluted lectin. After incubation for 1.5-2 h, the... 

Mix equal volumes of the cell sample and the erythrocyte suspension. 

FIGURE 2. Uptake of PDA by erythrocytes of several species. Erythrocyte suspensions (lO cells/ml) were exposed for 10 min to graded concentrations of PDA and centrifuged, and the... 

Corry WD, Jackson LJ, Seaman GV. Action of hydroxyethyl starch on the flow properties of human erythrocyte suspensions. Biorheology 1983 20(5) 705-17. 

Estimate hemoglobin content spectrophotometrically at 412 nm. The reciprocal dilution of the toxin hemolyzing 50% of the erythrocyte suspension at 37 °C within 40 min is taken as the number of hemolytic units per milliliter of the undiluted toxin solution. 

McGhee, R., and Trager, W. (1950). The cultivation of Plasmodium lophurae in vitro in chicken erythrocyte suspensions and the effects of some constituents of the culture medium upon its growth and reproduction. ]. Parasitol. 36,123-127. 

It should be stressed that due to very high values of buffer-membrane partition coefficients and low CMC values, the effect of resorcinolic lipids injected into the external medium is different from the effect observed when they were present internally in the membrane. For instance, the same homologues that are highly hemolytic when injected into erythrocyte suspension are not lytic when injected in the form of phosphatidylcholine-resorcinolic lipid liposomes, which indicates that direct exchange of resorcinolic lipids between membranes is limited. 

A stopped-flow rapid-reaction apparatus was used to measure the time course of pH changes in human erythrocyte suspensions. In one set of experiments a red cell suspension at pH 72 was mixed with an isotonic saline solution whose pH had been adjusted to a value between 2.1 and 10.4. Analysis of the results enabled computation of erythrocyte hydroxyl ion permeability as a function of pH. Further experiments were then performed in which erythrocyte suspensions at low pco2 were mixed with bicarbonate solutions at high pco2- Analysis revealed that C02 equilibrium in the mixture was reached quickly, but pH equilibrium was delayed. Evaluation of the results indicates that variation in red cell OH permeability with pH is not compatible with a simple fixed-charge hypothesis of membrane permselectivity, and the uncatalyzed hydration-dehydration of C02 in extracellular fluid is required to produce pH equilibration after blood-gas exchange. 

Figure 9. Computed curves of pC02 vs. time after mixing equal volumes of a 5% erythrocyte suspension (pco 0) and standard buffer solution (Pco2 65 torr) at 37°C (15)...

The most commonly applied methods for the analysis of polyamines in erythrocytes make use of amino acid analyzers and HPLC techniques. A capillary gas chromatographic method with nitrogen-phosphorous detection was applied to the simultaneous determination of 1,3-diaminopropane, putrescine, cadaverine (Cad), spermidine (Sd), and spermine (Sp) in human erythrocytes. Blood samples, collected by venipuncture into EDTA containing Venoject tubes, were subjected to the removal of plasma by centrifugation and erythrocytes were washed three times with two volumes of 0.9% NaCl. The stability of polyamines in erythrocyte suspensions was also investigated. Quantification of polyamines was done by comparing the peak-area ratio of each analyte and its internal standard with that of the standard. The polyamine samples were eluted with 0.1 M hydrochloric acid solutions. The eluate was evaporated to dryness at 120°C under a stream of air and 200 each of acetonitrile and heptafluorobutyric anhydride were added. The isolation of derivatives... 

The toxic activity is easily released from K. veneficum cells either by filtration and/or by centrifugation [139] (see Figure 33.6). However, under normal conditions greater than 90% of the toxin is cell bound [161]. A hemolytic assay based on the lysis of fish erythrocytes is routinely utilized to screen for karlotoxin in Karlodinium species [139]. Cultures and culture fractions that are positive in the hemolytic assay are tested further using assays for ichthyotoxicity and cytotoxicity as described above for Pfiesteria toxins. Erythrocyte suspensions are prepared as described in Edvardsen et al. 

Erythrocyte suspensions (20 % hematocrit) of 3 types were prepared with 2mM of ZnS04 with 10 pM of lead acetate, and the blank sample. The suspensions were fixed by two-perent solution of glutaraldehyde and were used for preparation of samples to AFM analysis. 

A major drawback of j8-CyD for parenteral use is its hemolytic effects due to its interaction with red blood cells, as demonstrated in vitro on erythrocyte suspensions. Studies performed on whole blood and erythrocyte suspension samples show that in all concentrations studied amphiphilic j8-CyD nanospheres are less hemolytic than native j8-CyD, because of their hydrophobic substituents [56]. Besides the hy-drophobicity, the self-assembly of amphiphilic j8-CyDs in the form of nanospheres is also believed to reduce the interaction and direct contact of CyD with red blood cells. 

Cokelet, G.R. andMeiselman,H.J. 1968. Rheological comparison of hemoglobin solutions and erythrocyte suspensions. Science 162 275-277. 

A different kind of support for Frohlich ideas has been brought by experiments on rouleaux formation in erythrocyte suspensions/ These experiments show that selective long-range interactions appear among metabolically active red blood cells. The connection between the experimental outcome and Frohlich theory has been recently discussed. 

One characteristic of many (but not all) saponins is their capacity to rupture erythrocytes (red blood corpuscles). By measuring the change in absorbance of the supernatant of an erythrocyte suspension after hemolysis, the saponin content can be calculated. Various amounts of the saponin-containing product or extract are mixed with a suspension of washed erythrocytes in isotonic buffer at pH 7.4. After 24 h, the mixture is centrifuged and hemolysis is indicated by the presence of hemoglobin (red) in the supernatant. In the European Pharmacopoeia, the quantity in milliliters of ox blood (diluted 1 50) that is totally hydrolysed by 1 g of test substance is measured. As a standard, the saponin mixture from the roots of Gypsophila paniculata (Caryophyllaceae) has by definition an activity of 30 000 units. 

Dilute the erythrocyte pellet 1 40 in PBS, which gives an about 2.5% erythrocyte suspension. 

Diepenhorst P. Removal of leucocytes from whole blood and erythrocyte suspensions by filtration through cotton wool (V). Vox Sang 1975 29(1) 15—22. 

If the test compounds inhibit virus replication and thus viral titre, the HA value will be reduced. HA inhibition assay is employed to test the effect of the test compounds in virus adsorption to target cells. Compound solutions (25 pi) with twofold serial dilution with PBS are mixed with equal volume of influenza virus solution (200HAU/25 pi). After incubation for 30 min at room tanperature, 50 pi of the solution is mixed with equal volume of 1% chicken erythrocyte suspension and incubated for 30 min at room temperature (Song et al. 2005). 

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