Glutaraldehyde-fixed Red Blood Cells Research Articles

Self-aligned sequential lateral field non-uniformities over channel depth for high throughput dielectrophoretic cell deflection.

Dielectrophoresis (DEP) enables the separation of cells based on subtle subcellular phenotypic differences by controlling the frequency of the applied field. However, current electrode-based geometries extend over a limited depth of the sample channel, thereby reducing the throughput of the manipulated sample (sub-μL min-1 flow rates and <105 cells per mL). We present a flow through device with self-aligned sequential field non-uniformities extending laterally across the sample channel width (100 μm) that are created by metal patterned over the entire depth (50 μm) of the sample channel sidewall using a single lithography step. This enables single-cell streamlines to undergo progressive DEP deflection with minimal dependence on the cell starting position, its orientation versus the field and intercellular interactions. Phenotype-specific cell separation is validated (>μL min-1 flow and >106 cells per mL) using heterogeneous samples of healthy and glutaraldehyde-fixed red blood cells, with single-cell impedance cytometry showing that the DEP collected fractions are intact and exhibit electrical opacity differences consistent with their capacitance-based DEP crossover frequency. This geometry can address the vision of an "all electric" selective cell isolation and cytometry system for quantifying phenotypic heterogeneity of cellular systems.

Finite element analysis of the lift on a slightly deformable and freely rotating and translating cylinder in two-dimensional channel flow.

Motivated by the lateral migration phenomena of fresh and glutaraldehyde-fixed red blood cells in a field flow fractionation (FFF) separation system, we studied the transverse hydrodynamic lift on a slightly flexible cylinder in a two-dimensional channel flow. The finite element method was used to analyze the flow field with the cylinder at different transverse locations in the channel. The shape of the cylinder was determined by the pressure on the surface of the cylinder from the flow field solution and by the internal elastic stress. The cylinder deformation and the flow field were solved simultaneously. The transverse lift exerted on the cylinder was then calculated. The axial and angular speed of the cylinder were iterated such that the drag and torque on the cylinder were nulled to represent a freely translating and rotating state. The results showed that the transverse lift on a deformable cylinder increased greatly and the equilibrium position moved closer to the center of the channel compared to a rigid cylinder. Also, with the same elastic modulus but a higher flow rate, a larger deformation and higher equilibrium location were found. The maximum deformation of the cylinder occurred when the cylinder was closest to the wall where a larger shear rate existed. The numerical results and experimental studies are discussed.

Low-frequency dispersion of surface conducting particles as measured by means of electrorotation

Electrorotation of glutaraldehyde-fixed red blood cells (RBCs) has been investigated in the frequency range from 16 Hz to 30 MHz. Special emphasis has been given to the low-frequency range (16 Hz–2 kHz). In this region, a distinctly new type of co-field rotation peak was observed. Native RBCs also show low-frequency rotation, but much less pronounced. The low-frequency rotation is nonmonotonous with regard to conductivity. With increasing external conductivity, the low-frequency rotation speed reaches a maximum of around 3.5 mS/m and decreases again when the external conductivity is increased further. The low-frequency co-field rotation peak was observed between 30 Hz and 70 Hz except at very low conductivity almost independent of electrolyte concentration. Decreasing surface charge density by means of neuraminidase led to a reduction of the rotation speed in the low-frequency range. The position of the peak remained unaffected by changes of the surface charge density. These experimental results are not consistent with the typical Pauli–Schwan- and Maxwell–Wagner-dispersion. They rather suggest the presence of a concentration polarization mechanism responsible for the low-frequency electrorotation peak.

Organ targeting in vivo using phage display peptide libraries.

Preferential homing of tumour cells and leukocytes to specific organs indicates that tissues carry unique marker molecules accessible to circulating cells. Organ-selective address molecules on endothelial surfaces have been identified for lymphocyte homing to various lymphoid organs and to tissues undergoing inflammation, and an endothelial marker responsible for tumour homing to the lungs has also been identified. Here we report a new approach to studying organ-selective targeting based on in vivo screening of random peptide sequences. Peptides capable of mediating selective localization of phage to brain and kidney blood vessels were identified, and showed up to 13-fold selectivity for these organs. One of the peptides displayed by the brain-localizing phage was synthesized and shown to specifically inhibit the localization of the homologous phage into the brain. When coated onto glutaraldehyde-fixed red blood cells, the peptide caused selective localization of intravenously injected cells into the brain. These peptide sequences represent the first step towards identifying selective endothelial markers, which may be useful in targeting cells, drugs and genes into selected tissues.

Phagocytosis and ATP levels in alveolar macrophages during acute hypoxia.

Pulmonary alveolar macrophages (PAM) function as phagocytes of inhaled particulate matter and microorganisms at the air-tissue interface of lung alveoli. Changes in cellular ATP concentrations ([ATP]) and phagocytic function during acute hypoxia may be important in conditions associated with low alveolar O2. We proposed that acute hypoxia would decrease phagocytosis and reduce [ATP] in freshly isolated PAM. Phagocytic function (fluorescent microscopic technique determining percent phagocytosis in live cells) was monitored by recording uptake and retention of glutaraldehyde-fixed red blood cells (GRBC) in isolated rabbit PAM during acute incubations in air (20% O2) or hypoxia (1.7% O2). Macrophage [ATP] were determined spectrophotometrically. Acute hypoxia for 30 to 150 min decreased phagocytic function 30 to 56% in PAM without significantly affecting cell adherence and viability. Pre-exposure of PAM to hypoxia before addition of GRBC resulted in an even greater reduction in phagocytosis (97% decrease by 30 min), and recovery of phagocytic function occurred 60 to 90 min after returning PAM to air. The cellular retention of phagocytosed GRBC (percentage of PAM with GRBC and number of GRBC/PAM) was reduced 30% by 1 h of hypoxia. Compared with [ATP] of PAM in air, [ATP] of PAM exposed to hypoxia were reduced 55 and 35% at 30 and 60 min, respectively. Compared with [ATP] of cells with GRBC in air at 0 and 30 min, PAM with GRBC in hypoxia for 30 min had, respectively, 61 and 40% lower [ATP]. By 60 min with GRBC, PAM [ATP] in air and hypoxia were similar but were 50% lower than [ATP] at time 0.(ABSTRACT TRUNCATED AT 250 WORDS)

Microspectrophotometric and scanning microphotometric studies of carp (Cyprinus carpio L.) erythrocytes.

Carp (Cyprinus carpio) hemoglobin readily autoxidizes in blood smears. Quantification of Soret-band absorbance in individual erythrocytes by means of scanning cytophotometry therefore requires more elaborate methods of preparation of blood samples. Of the fixatives that have been tested, suspension of whole blood in isotonic salt solutions containing glutaraldehyde was most suitable. Glutaraldehyde-fixed red blood cells are totally resistant to hemolysis. In the course of fixation, hemoglobin is transformed to methemoglobin. Spectrophotometry indicated extensive similarities between glutaraldehyde-fixed carp methemoglobin and human methemoglobin. In aqueous solutions, the intensity of the Soret-peak was pH-dependent. The allosteric modifier organic polyphosphate caused an R----T transition, resulting in increased molar extinctions. Dried preparations showed Soret-spectra that were not influenced from either pH or organic polyphosphate concentration of the aqueous suspensions in which the erythrocytes had been stored. The same was true for slide preparations of cyanomethemoglobin, easily derived from methemoglobin on addition of potassium cyanide. In the absence of oxygen fresh blood cells from carp slowly transform their hemoglobin into deoxyhemoglobin. Spectra of the intermediate stages of deoxygenation, Hb4(O2)3, Hb4(O2)2 and Hb4(O2), as well as mixtures of these intermediates, could be monitored.

A fluorescence quenching technique using trypan blue to differentiate between attached and ingested glutaraldehyde-fixed red blood cells in phagocytosing murine macrophages

A fluorescence quenching method, using trypan blue, is described for quantifying the ingestion of either glutaraldehyde-fixed sheep red blood cells or antibody coated glutaraldehyde-fixed sheep red blood cells by murine peritoneal macrophages. This method is based on the observations that glutaraldehyde-fixed red blood cells fluoresce at about 585 nm when excited at 490 nm and that when trypan blue is in intimate contact with fixed red blood cells, the fluorescence is converted from a chartreuse to a red color. Thus, the ingestion of fixed erythrocytes by murine macrophages can be monitored by fluorescence microscopy after the addition of 1 mg/ml of trypan blue at the end of the assay. Extracellular glutaraldehyde-fixed red blood cells fluoresce a red color whereas the intracellular particles continue to fluoresce a chartreuse color. This method offers a simple and convenient technique for rapidly distinguishing between intracellular and extracellular glutaraldehyde-fixed red blood cells in macrophages.

Red blood cell adhesion. III. Analysis of forces.

The results of experiments on the adhesion of glutaraldehyde-fixed red blood cells to both polarizable metal/saline and liquid hexadecane/saline interfaces have been analysed in terms of physical forces. The results show that the electrostatic repulsions sufficient to prevent adhesion to these test surfaces are remarkably similar, and that a force-balance condition is predicted at cell-substratum separations similar to or approximately 100 nm in 0.4 mM NaCl as found by interferometry. From the repulsive force the size of the attractive force can be found. If this is viewed as an electrodynamic attraction, the force coefficient is found to lie between 5 and 8 x 10(-14) erg (5 and 8 x 10(-21)J), a range in reasonable correspondence with measurements in physical systems.

Fusion of macrophages following simultaneous attempted phagocytosis of glutaraldehyde-fixed red cells.

A model for the fusion of macrophages in areas of chronic inflammation in vivo has been presented which is derived from the associations of macrophage fusion in vivo and in vitro. It is suggested that in granulomata, where macrophages are closely packed in the presence of endocytogenic material, this material may on occasion attach simultaneously to more than one macrophage. Ensuing endosome margin formation may then lead to the endosome margins of one macrophage fusing with those of the other. By causing macrophages to phagocytose glutaraldehyde-fixed red blood cells in vitro under circumstances which more closely reflect in-vivo phagocytosis than the phagocytosis of the red cells by macrophages in a glass adherent monolayer, it has been shown that simultaneous attachment leads to macrophage fusion. It is suggested that giant cell formation in vivo in granulomata results from the process of simultaneous attempted endocytosis.