Search

We previously developed a Deterministic Lateral Displacement (DLD) microfluidic method in silicon to separate cells of various sizes from blood (Davis et al., Proc Natl Acad Sci 2006;103:14779-14784; Huang et al., Science 2004;304:987-990). Here, we present the reduction-to-practice of this technology with a commercially produced, high precision plastic microfluidic chip-based device designed for automated preparation of human leukocytes (white blood cells; WBCs) for flow cytometry, without centrifugation or manual handling of samples. After a human blood sample was incubated with fluorochrome-conjugated monoclonal antibodies (mAbs), the mixture was input to a DLD microfluidic chip (microchip) where it was driven through a micropost array designed to deflect WBCs via DLD on the basis of cell size from the Input flow stream into a buffer stream, thus separating WBCs and any larger cells from smaller cells and particles and washing them simultaneously. We developed a microfluidic cell processing protocol that recovered 88% (average) of input WBCs and removed 99.985% (average) of Input erythrocytes (red blood cells) and >99% of unbound mAb in 18 min (average). Flow cytometric evaluation of the microchip Product, with no further processing, lysis or centrifugation, revealed excellent forward and side light scattering and fluorescence characteristics of immunolabeled WBCs. These results indicate that cost-effective plastic DLD microchips can speed and automate leukocyte processing for high quality flow cytometry analysis, and suggest their utility for multiple other research and clinical applications involving enrichment or depletion of common or rare cell types from blood or tissue samples. © 2016 International Society for Advancement of Cytometry.

Antibody to HCV core and NS3 was quantified by using a microsphere immunoassay and flow cytometry. Antibody to core and NS3 was elevated in the 85 seropositive blood donors tested. The amount of either antibody varied over two logs although greater variation was seen with the antibody to NS3 than was seen with antibody to core. In three documented acute HCV cases, the microsphere assay detected antibody prior to antibody detection using the reference methods. Twenty donor samples were indeterminate by the reference methods: 45% of these were indeterminate with the microsphere assay while 25% were negative and 30% were positive. As compared to enzyme immunoassay the microsphere assay showed a 5-fold increase in sensitivity. The microsphere assay demonstrated increased sensitivity for the quantification of specific antibody to HCV core and NS3 and was useful in resolving a significant proportion of indeterminate samples. Cytometry 29:106‐112, 1997. r 1997 Wiley-Liss, Inc.

The ability to conjugate fluorescent dyes of distinct colors to specific probes has permitted analysis of cells present within complex and heterogeneous populations. General considerations for selection of appropriate combinations of fluorescent dyes for immunofluorescence, conjugation of fluorochromes with protein probes, and quantitation of fluorescence are reviewed.

A method for the absolute quantitation of particle fluorescence on a digital microscope has been characterized using commonly available imaging software. The method uses solid glass beads in a known concentration of dye solution. The dye volume displaced by the beads and the drop in fluorescence intensity are measured. The intensity drop associated with the number of dye molecules displaced is given by the negative integral of the dark spots (beads) in the image. As expected, results show a linear correlation between intensity drop and bead volume for a given concentration and a linear correlation between intensity drop over bead volume and concentration of dye

The use of protein-based biological pigments in combination with low molecular weight organic dye molecules allows the simultaneous detection of up to six independent components of a cell surface by antibody dependent fluorescence (immunofluorescence) excited with a single 488nm laser line. This paper describes the state of the art in multi-color immunofluorescence with 488nm Ar-ion laser excitation. It provides the data to design systems for specialized applications.

Methods are described to classify nucleotide binding sites of the mitochondrial coupling factor F1 from yeast on the basis of their affinities and stability properties. High affinity sites or states for ATP and related adenine analogs and low affinity sites or states which bind a broad range of different nucleotide triphosphates are found. The results are discussed in terms of a two site, two cycle scheme, where binding of nucleotide at one site facilitates the release of nucleotide at a second site.

One of the problems of chemical evolution on the primitive Earth is the question of the possibility of a non-enzymatic spontaneous condensation o f phosphoric acid and hydrogen phosphates to yield polyphosphoric acids and polyphosphates in aqueous system s, by means of which phosphorus might have entered into early metabolisms. The extra- or intra-cellular formation of magnesium diphosphate under geologically plausible hydrothermal conditions from either magnesium hydrogen phosphates or calcium phosphates in media has been demonstrated.

In common with the F1-ATPase from other sources, yeast mitochondrial F1-ATPase was inhibited by 4-chloro-7-nitrobenzofurazan. Total inhibition of the F1-ATPase activity was compatible with the modification of a single tyrosine residue per F1-ATPase molecule. Radioactive labelling experiments localized this modification on a β-subunit. The inactive modified enzyme retained the capacity to bind the photoaffinity label 8-azido-1,N6-etheno-ATP, which has previously been shown to bind nucleotide sites of low affinity. As well, the inactive modified enzyme bound MgATP with high affinity, yielding a Kd of 14 μM. The results are consistent with the hypothesis of alternating, or cooperative, site catalysis by F1-ATPase.

Mitochondrial ATP synthase from yeast is an oligomeric enzyme consisting of a hydrophobic membrane part (Fe) and a hydrophilic part (Fr). Fr is composed of 5 different polypeptide chains (o to c) [Il. Lowand high-affinity nucleotide diand triphosphate binding sites have been characterized on the enzyme from various sources by different kinetic techniques [2,3]. Alternatively, binding sites have been located on specific subunits by photoaffinity labelling [4-81. Recently, the development of a fluorescent photoaffinity label (8-azido-I ,Ne-ethenoATP) was reported [9], which might allow the identification of a binding site with respect to the hydrolytic or synthetic function of Fr . We have therefore investigated the interaction of this compound with yeast Fr . It will be shown that the label interacts with a low-affinity nucleotide binding site, and that upon photolabelling it is bound to the P-subunit.

A vital, nucleic acid stain (LDS-751) was used to discriminate intact from damaged cells in a flow cytometer even after the samples had been fixed with paraformaldehyde. Three major cell populations with different fluorescence properties with LDS-751 were found in the fixed samples. Cells not staining or only dimly staining with LDS-751 were identified as erythrocytes and platelets, respectively. Cells staining with intermediate amounts of LDS-751 were found to be intact cells, while cells intensively stained were identified as damaged cells. Confirmation of the identity of the populations was obtained by light microscopic examination of the sorted populations and by correlating the fluorescent signals of FDA and LDS-751 in nonfixed cell preparations. Agglutinated cells could also be identified by the increased fluorescent signal in the LDS-751 channel as compared with single cells. The spectral properties of this dye permit excitation at 488 nm with emission in the far red portion of the spectrum. This allowed two-color immunofluorescence to be combined with the intact/damaged cell discrimination on fixed samples. Therefore, intact single cells could be distinguished during flow cytometric analysis, increasing the accuracy of the immunofluorescence measurements. The visualization of the multidimensional data was facilitated using color to discriminate cell populations depicted in multiple perspectives.

Received 6 June 1977 1. Introduction Soluble ATPase of mitochondria is now an accepted component of the enzyme system catalyzing oxidative phosphorylation. According to the hypothesis of Boyer and Slater [ 1,2] a conformational transition of the enzyme protein is expected to be involved in ATP synthesis and the kinetic resolution of the ATPase reaction should give further insight into the mechanism of energy conservation in oxidative phosphorylation. The high purity of an ATPase preparation obtained in this laboratory [3] allowed to analyze the transient state kinetics of the hydrolytic reaction catalyzed by the enzyme. We here report that the catalytic process can be resolved into two steps, one of which is a slow conformational transition with a time constant of the order of 10 see-’ followed by a steady state hydrolysis with a turnover number of 200 set-‘. A preliminary report of this work was given elsewhere [4]. 2. Material and methods The soluble ATPase was prepared according to [3]. The specific activity of the enzyme was about 150 U/mg. The turnover number is given under the assumption of three active sites on the enzyme. The overall activity was determined in an ATP regenerating assay system as given elsewhere [ 51. For stopped flow experiments the enzyme protein was obtained from a stock solution and transferred to the appropriate buffer by spinning down the ammonium sulfate suspension of the enzyme, dissolving the pellet in the desired buffer and passing it through a Sephadex G-50 column preequilibrated with the same buffer. The enzyme was used immediately after this treatment in order to avoid significant activity loss occurring in the complete absence of ATP. Protein concentrations are based on a molecular weight of 400 000 [3]. ATP was obtained from Boehringer, Mannheim, E-ATP from Sigma and AMP-PNP (&y-imidoadenosine-5’-tri- phosphate) as well as phenolred from Serva, Heidelberg. All other chemicals were of the purest grade commercially available. The concentrations of ATP, E-ATP, AMP-PNP and phenolred were determined by absorbancy measurements in a Zeiss PMQ II photometer. Stopped flow experiments using phenolred as indicator were carried out with an instrument described elsewhere [6]. The proton release was evaluated by converting the obtained absorbancy changes of the indicator at 560 nm to proton concentrations liberated in the reaction using buffer-capacity of the system. The results were consistent with the end-point of the reaction as calculated from the total amount of ATP present in the reaction mixture. Experiments using the fluorescence change of E-ATP Mn(II) were performed with a Durrum stopped flow instrument connected to a Digital Storage oscilloscope (Nicolet Instrument Corporation Model 1074) allowing signal averaging. Here, the quenching effect of Mn’+- ions upon the fluorescence of E-ATP allows one to follow the reaction [7]. ATP forms a more stable complex with Mn”-ions than ADP, thus changing the amount of nucleotide present as manganese complex at constant manganese-concentrations.

A rapid sensitive method for the determination of Plasmodium falciparum in in vitro culture is presented. The technique employs a fluorescent flow cytometer equipped with a 15-mwatt argon laser that emits light at 488 nm and a membrane-permeable fluorochrome thiazole orange (TO) that stains RNA. Parasitized red cells are stained by suspending them in 1 ml of phosphate-buffered saline (PBS) containing 10(-5) M of TO and incubating this mixture for 15 min in the dark at room temperature. The stained cells may be analyzed fresh or after fixation with 1% paraformaldehyde/PBS or 0.25% glutaraldehyde/PBS. Alternatively the cells may be fixed first and then stained. There is excellent correspondence between the number of fluorescent-labeled parasitized red cells and Giemsa-stained cells.

The paramagnetic resonance spectra of two spin-labels, 2,2,6,6-tetramethylpiperadinyl-1-oxy and a head-group spin-labeled phosphatidylethanolamine (L-alpha-dipalmitoylphosphatidyl-N-ethanolamine), have been used to study solid-liquid and liquid-liquid phase separations in binary mixtures of dimyristoylphosphatidylcholine and cholesterol. A quantitative analysis of these resonance spectra supports the view that at temperatures below theta m, the chain-melting temperature of the phospholipid, and at cholesterol mole fractions Xc less than 0.2, these mixtures consist of two phases, a solid phase of essentially pure dimyristoylphosphatidylcholine and a fluid phase having a mole fraction of cholesterol equal to 0.2. The spin-label data also provide evidence for fluid-fluid immiscibility in the bilayer membrane at temperatures above the chain melting transition temperature of dimyristoylphosphatidylcholine.

Immunochemical assays are based on the fact that the mammalian immune system forms protein molecules, which bind with high affinity to an antigen molecule. All kinds of molecules can be antigen molecules, and the specificity of these proteins, called antibodies, is rather high. When an animal is injected (immunized) with an antigen, the resulting immune response leads to the generation of a variety of antibody molecules against the antigen, a serum. The composition of the serum depends on the history of the animal. Therefore it is not always easy to reproduce the exact antibody mixture of a serum in different immunizations. These antibody mixtures are very specific, because they recognize a number of different sites or epitopes on the antigen molecule. Each of these antibody molecules is made by one B-cell type. These B-cells can be isolated and grown. A colony which is generated from a single B-cell creates one kind of antibody molecule, a monoclonal antibody. Monoclonal antibodies bind or recognize one epitope on an antigen molecule. As a consequence, monoclonal antibodies bind to any molecule which shows this epitope or molecular geometry. Once a B-cell culture is established, monoclonal antibodies can be reproduced in large quantities.