Hank's Buffered Salt Solution Research Articles

Spectral characterization of the voltage-sensitive dye di-4-ANEPPDHQ applied to probing live primary and immortalized neurons

Spectral properties of a recently developed voltage-sensitive dye, di-4-ANEPPDHQ, were characterized as the dye was dissolved in the solvent dimethyl sulfoxide as the stock solution, in Hank's buffered salt solution as the staining solution, and bound to the plasma membrane of primary rat hippocampal neurons and immortalized mouse hypothalamic neurons (GT1-7) in vitro. Their dependence on the local chemical and electrical environment of dye molecules was determined. The excitation and emission peaks are 479 nm and 570 nm for the stained primary neurons, and 476 nm and 585 nm for the stained immortalized neurons. The excitation and emission bands of the stained GT1-7 neurons, defined as 50% peak intensity, are 429-516 nm and 544-648 nm, respectively.

No evidence of myocardial restoration following transplantation of mononuclear bone marrow cells in coronary bypass grafting surgery patients based upon cardiac SPECT and 18F-PET

BackgroundWe tested the hypothesis, that intramyocardial injection of mononuclear bone marrow cells combined with coronary artery bypass grafting (CABG) surgery improves tissue viability or function in infarct regions with non-viable myocardium as assessed by nuclear imaging techniques.MethodsThus far, 7 patients (60 ± 10 [SD] years) undergoing elective CABG surgery after a myocardial infarction were included in this study. Prior to sternotomy, bone marrow was harvested by sternal puncture. Mononuclear bone marrow cells were isolated by gradient centrifugation and resuspended in 2 ml volume of Hank's buffered salt solution. At the end of CABG surgery 10 injections of 0.2 ml each were applied to the core area and borderzones of the infarct. Global and regional perfusion and viability were evaluated by ECG-gated 99mTc-tetrofosmin myocardial single-photon emission computed tomograph (SPECT) imaging and 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) in all study patients < 6 days before and 3 months after the intervention.ResultsNon-viable segments indicating transmural defects were identified in 5 patients. Two patients were found to have non-transmural defects before surgery. Concomitant surgical revascularisation and bone marrow cell injection was performed in all patients without major complications. The median total injected mononuclear cell number was 7.0 × 107 (range: 0.8–20.4). At 3 months 99mTc-tetrofosmin SPECT and 18F-FDG-PET scanning showed in 5 patients (transmural defect n = 4; non-transmural defect n = 1) no change in myocardial viability and in two patients (transmural defect n = 1, non-transmural defect n = 1) enhanced myocardial viability by 75%. Overall, global and regional LV ejection fraction was not significantly increased after surgery compared with the preoperative value.ConclusionIn CABG surgery patients with non-viable segments the concurrent use of intramyocardial cell transfer did not show any clear improvement in tissue viability or function by means of non-invasive bioimaging techniques.

Supramolecular Fluorophores for Biological Studies: Phenylene Vinylene-Amino Acid Amphiphiles

We report here on a family of self-assembling fluorescent organic amphiphiles with a biomolecular L-lysine hydrophile and a photonically active phenylene vinylene hydrophobe. Unlike conventional amphiphiles, these segmented dendrimers feature a rigid, branched hydrophobe, and have packing characteristics controlled by the ratio of cross-sectional areas of the hydrophobe and hydrophile. In dilute solution, the amphiphiles form supramolecular aggregates, which are easily taken in by cells through an endocytic pathway, and have no discernible effect on cell proliferation or morphology. An analogous pyrene-based amphiphile was cytotoxic, suggesting that cell survival may be linked either to the self-assembling nature of the amphiphiles, or to the specific properties of the phenylene vinylene segment. The combination of photonic and biological components in these amphiphiles provides great potential for applications in sensing or delivery of molecules to intracellular targets.

Treatment of Retinal Breaks with Autologous Serum in an Experimental Model

Purpose: The standard treatment for retinal breaks is thermal adhesion. Breaks in the posterior pole (i.e., macular holes) recently have been treated using vitrectomy and the recombinant cytokine transforming growth factor-beta. This has been shown to achieve closure of the retinal breaks by stimulating localized fibrocellular proliferation. Serum has been shown to contain chemoattractants and mitogens for many types of cells. The authors studied the clinical and histologic effect of autologous serum application to retinal breaks in an experimental model. Method: Twenty-four rabbits underwent pars plana lensectomy, vitrectomy, retinectomy, fluid-air exchange, application of test solution (12 with Hank's buffered salt solution and 12 with autologous serum), and air-gas exchange. Clinical examination with indirect ophthalmoscopy was performed, and animals were killed 5, 14, and 28 days after treatment. Tissue sections through the retinectomy were studied by light microscopy, electron microscopy, and immunocytochemistry. Results: None of the serum-treated eyes showed retinal detachment at the site of the retinectomy by evaluation with indirect ophthalmoscopy at each of the time points. In contrast, in control eyes retinal detachment developed at the retinectomy site from 0% at day 5 to 50% at day 14 and 75% at day 28. By light microscopy, serum-treated eyes contained multilayers of fibroblast-like cells adhering the retinectomy edges to the underlying retinal pigment epithelium and choroid. The control eyes had nonadherent retinal edges at the retinectomy site with little sign of fibrocellular response. Results were confirmed by electron microscopy. The fibroblast-like cells by immunocytochemistry contained vimentin, cytokeratin 18, and jor glial fibrillary acidic protein. Conclusion: This study suggests that serum induces a localized fibrocellular response at the retinectomy edges compared with control eyes. This response, characterized by light microscopy, electron microscopy, and immunocytochemistry, appears to involve a mixed population of glial, retinal pigment epithelial, and/or fibroblastic cells. These cells seem to enhance adhesion and subsequent reattachment of the edges of the retinectomies at the time points studied when compared with controls.

Relationship of actin filament assembly to clearance of fibrinogen gold, GPIIb-IIIa complexes on spread platelets.

The present study has evaluated the influence of high concentrations of cytochalasins B and E on the detergent-resistant actin levels in fully spread platelets by PAGE gel electrophoresis, and the effects of the two inhibitors of new actin filament assembly on translocation of fibrinogen gold (Fgn/Au) labelled GPIIb-IIIa receptors on the surface-activated cells. Concentrations of 10(-4) M and 10(-5) M cytochalasin B and E reduced detergent-resistant actin in fully spread platelets to levels present in resting discoid platelets in suspension. Despite reduction of actin filaments to levels in resting cells, cytochalasin B did not prevent translocation of Fgn/Au from platelet margins into channels of the open canalicular system (OCS). Similar concentrations of cytochalasin E completely blocked translocation of receptor-ligand complexes and produced a patching phenomenon not observed in previous studies. Rinsing of the spread cells to remove cytochalasin, followed by incubation of the treated platelets in Hank's buffered salt solution (HBSS) restored levels of detergent-resistant actin to those found in untreated, spread platelets. Resting grids of 10(-5) M cytochalasin E-treated platelets on drops of HBSS for 15 min restored their ability to clear FGN/Au linked to GPIIb-IIIa from exposed surfaces to the OCS, but 10(-4) M cytochalasin E-treated cells remained anergic after incubation on drops of HBSS. Thus a fully assembled cytoplasmic actin filament cytoskeleton does not appear to be essential for translocating receptor-ligand complexes on the platelet surface to the OCS, nor does its presence guarantee that the ability to clear GPIIb-IIIa receptors will be restored.

Treatment of Retinal Breaks with Autologous Serum in an Experimental Model

The standard treatment for retinal breaks is thermal adhesion. Breaks in the posterior pole (i.e., macular holes) recently have been treated using vitrectomy and the recombinant cytokine transforming growth factor-beta. This has been shown to achieve closure of the retinal breaks by stimulating localized fibrocellular proliferation. Serum has been shown to contain chemoattractants and mitogens for many types of cells. The authors studied the clinical and histologic effect of autologous serum application to retinal breaks in an experimental model. Twenty-four rabbits underwent pars plana lensectomy, vitrectomy, retinectomy, fluid-air exchange, application of test solution (12 with Hank's buffered salt solution and 12 with autologous serum), and air-gas exchange. Clinical examination with indirect ophthalmoscopy was performed, and animals were killed 5, 14, and 28 days after treatment. Tissue sections through the retinectomy were studied by light microscopy, electron microscopy, and immunocytochemistry. None of the serum-treated eyes showed retinal detachment at the site of the retinectomy by evaluation with indirect ophthalmoscopy at each of the time points. In contrast, in control eyes retinal detachment developed at the retinectomy site from 0% at day 5 to 50% at day 14 and 75% at day 28. By light microscopy, serum-treated eyes contained multilayers of fibroblast-like cells adhering the retinectomy edges to the underlying retinal pigment epithelium and choroid. The control eyes had nonadherent retinal edges at the retinectomy site with little sign of fibrocellular response. Results were confirmed by electron microscopy. The fibroblast-like cells by immunocytochemistry contained vimentin, cytokeratin 18, and/or glial fibrillary acidic protein. This study suggests that serum induces a localized fibrocellular response at the retinectomy edges compared with control eyes. This response, characterized by light microscopy, electron microscopy, and immunocytochemistry, appears to involve a mixed population of glial, retinal pigment epithelial, and/or fibroblastic cells. These cells seem to enhance adhesion and subsequent reattachment of the edges of the retinectomies at the time points studied when compared with controls.

57 IN SITU FLUORESCENCE ANISOTROPY MEASUREMENTS IN CULTURED CELLS BY A COVERSLIP TECHNIQUE USING 1-(4-TRIMETHYL-AMMONIUMPHENYL) −6-PHENYL −1, 3, 5− HEXATRIENE (TMADPH)

Fluorescence anisotropy (rG) was determined in monolayers of cultured cells grown on glass coverslips under various conditions with 5μM TMA-DPH in Hank's buffered salt solution at 37°C. rG was inversely related to membrane fluidity and increased with density of cells and age in culture. rG (±SD) was different in confluent human fibroblasts (0.348±0.016), rat astrocytes (0.315 ±0.011) and rat Roc-1 hybridoma cells (oligodendrocytes × C6) (0.304 ±0.018). Environmental factors like temperature, Ca++ and osmolarity as well as X-ray irradiation modified anisotropy in human fibroblasts. rG decreased in absence of extracellular Ca++ (0.301±0,027) and under hypotonic conditions (30 mosmol mannitol reduced rG to 0.298 ±0.015). On the other hand rG was shown to increase at deeper temperatures and under hypertonic conditions (550 mosmol mannitol led to rG of 0.388±0.013). Conclusions: TMA-DPH is a marker of superficial layers of the membrane. Fluorescence anisotropy measurements in situ allow to study cellular and environmental factors in cultured cells and provide a sensitive method for detecting of possible genetic or acquired alterations of the membrane.

Dissolution of eosinophil granules in bronchial lavage obtained from allergic asthmatics during bronchoprovocation and seasonal exposure

Bronchial lavage was used to assess changes in cell populations from the airways of allergic asthmatic patients who were exposed seasonally to allergens or challenged with aerosolized allergen. Bronchoprovocation (BPC) was used to elicit early and late asthmatic responses (LAR), and bronchial lavage was performed at baseline out of season or at either 2, 6, or 24 hours after challenge, and during the season in five mildly symptomatic patients. Four normal subjects were challenged with allergen and lavaged either 2 or 6 hours later.Bronchial lavage fluid and cells from subsegmental bronchi were immediately passed through 2 layers of gauze and cells were washed once with Hank's buffered salt solution and gently centrifuged. Cyto-preps of lavage cells were prepared and examined by light, microscopy with a Wright-Giemsa stain. These cells were then mixed with 50% Percoll and centrifuged at 1500 RPM for 20 minutes. This procedure was adopted to concentrate small numbers of cells such as eosinophils and mast cells into a pellet which contained relatively few macrophages.

Lymphocyte response to phytohemagglutinin: intracellular volume and intracellular [K+

The effect of phytohemagglutinin (PHA) on lymphocytes was examined with respect to free intracellular water volume and intracellular [K+]. At a cell concentration of 30 X 10(6) lymphocytes/ml in modified Hank's Buffered Salt Solution (HBSS) in the presence of 10% human AB serum, addition of PHA at 3 mg/ml resulted in a 24-27% decrease in free intracellular water space within 30 to 60 minutes and a return to control level after three hours. A larger change in intracellular water (44%) was observed under similar conditions in the absence of serum. The absolute intracellular K+ content did not change after PHA addition, but the cell water volume decrease arising from PHA addition resulted in a 29% increase in intracellular [K+] at 60 minutes. The decrease in lymphocyte water volume induced by PHA was also observed for concanavalin A which stimulates lymphocyte proliferation, but not for wheat germ lectin, an agglutinating agent which is not mitogenic. Thus, volume regulation may be closely associated with the mitogenicity of these compounds.