An enzyme immunoassay for estimating small numbers of cells

In this report we describe an enzyme immunoassay for determination of cell number in small samples. The assay utilizes a commercially available monoclonal antibody that recognizes double- and single-stranded DNA and works with several different methods of cell fixation. DNA extraction is not required. Confluent, growth-arrested fibroblasts serve as standards. While most readily applicable to anchorage-dependent cells, addition of a centrifugation step allows the method to be applied also to anchorage-independent cells.     

A simple method for obtaining cell-specific cDNA from small numbers of growing root-hair cells in Arabidopsis thaliana

A simple and rapid method for cloning specific cDNAs from mRNA populations derived solely from small numbers of root-hair cells is described here. To identify genes expressed during the earliest visible stage of root-hair cell development, cell contents were aspirated from small numbers of Arabidopsis root-hair cells at or just before this stage. This material was used to make reusable solid-phase oligo-dT-primed cDNA libraries. To demonstrate that the libraries contained high quality longer cDNAs, a fragment located 2.7 kb from the 3' end of the cDNA of the single copy root-hair expressed gene RHD3 was cloned using a nested PCR strategy. This technique was also used to obtain novel gene expression information by cloning the full-length 0.85 kb cDNA of the Rop2 GTPase from this library. This approach offers a means of cloning larger cDNAs directly from small numbers of growing root-hair cells and, potentially, other epidermal cell types.     

A simple method for the assay of eight steroids in small volumes of plasma

A simple method is described for the simultaneous radioligand assay of four Δ⁵-3β-hydroxysteroids adjacent to one another on the biosynthetic pathway (pregnenolone [1], 17α-hydroxypregnenolone, dehydroepiandroste rone and 5-androsterone-3β,17β-diol), and their four Δ⁴-3keto products (progesterone, 17α-hydroxyprogesterone, 4-androstene-3, 17-dione and testosterone). Two plasma aliquots are extracted and fractionated each for four steroids and individual corrections are made for losses. For fractionation, maximum use is made of the high resolution and reproducibility of celite minicolumns, using propylene glycol as stationary phase, and a discontinuous gradient of ethyl acetate in iso-octane as mobile phase. The fractions are then assayed in the appropriate radioligand end-assay system. Each assay was finally validated by demonstrating coincidence of peaks of immuno- and radioactive steroid In extracts of female plasma. Results in pre-pubertal girls and women in the follicular phase of the menstrual cycle suggest that the major change in adrenal steroid production at puberty may be an increase in 17,20-desmolase activity. There appears to be little reversal of this change in adrenal function after ovariectomy.     

A simple and novel method for recovering adenovirus 41 in small volumes of source water

Aim: A new procedure was developed to recover adenovirus 41 in small volumes (1 l) of water samples based on adsorption, elution and evaporation. Methods and Results: One litre of source water seeded with adenovirus 41 was adjusted to pH 3·5 and filtered using a large pore size (8·0 μm) negatively charged membrane filter (SCWP, 47 mm diameter, made of mixed‐cellulose esters). Then, the filter was eluted using 4 ml of 1·5% beef extract plus 0·75% glycerol (pH 9·0). The eluate was reconcentrated to 0·1 ml or less volumes through evaporation assisted with air flow and heating at 55°C. Recovery of adenovirus 41 reached 55% under tested conditions and reduced filtration time by 85% in contrast to the widely used small pore size filter (0·45 μm pore size, 47 mm diameter). Reconcentration by evaporation achieved approx. 86·8% recovery from source water in approx. 1 h at no cost. Conclusion: The virus concentration method developed in this study is simple and cost‐effective and can be used to efficiently recover adenovirus 41 from turbid water samples. Significance and Impact of the Study: The procedure developed can be applied to detect adenovirus 41 in source water within hours of sampling. In addition, this is the first application of evaporation to concentrate viruses in water samples.     

A simple and fast extraction method for organochlorine pesticides and polychlorinated biphenyls in small volumes of avian serum

A solid-phase extraction (SPE) method was developed using 8M urea to desorb and extract organochlorine pesticides (OCs) and polychlorinated biphenyls (PCBs) from avian serum for analysis by capillary gas chromatography with electron capture detection (GC-ECD). The analytes were efficiently extracted from the denatured serum-lipoprotein-analyte complex by one passage through an Oasis((R)) hydrophilic-lipophilic-balanced (HLB) SPE cartridge. No further clean-up was necessary, the entire extraction procedure and GC-ECD analysis can be accomplished in less than 3h. Serum volumes ranged from 100 microL to 1 mL with absolute recoveries of 90-101% for PCBs and 74% to 101% for the OC pesticides.     

Freeze-fracture cytochemistry: a new method combining immunocytochemistry and enzyme cytochemistry on replicas.

We describe a new freeze-fracture cytochemical technique consisting of combined immunocytochemistry and enzyme cytochemistry. This technique reveals the relationship between molecules in biological membranes by double labeling with two different cytochemical markers (i.e., immunogold probes and cerium). In this method, antigens were detected with specific primary antibodies and appropriate secondary immunoprobes. Subsequently, alkaline phosphates activity was detected with cerium as the capture agent on the same replicas. Octyl-glucoside (OG) digestion before the cytochemical reactions was crucial to the success of this combined method. OG is an efficient detergent and OG digestion can preserve both immunocytochemical antigenicity and enzyme activity on replicas. As an initial examination, we applied this technique to the study of glycosyl-phosphatidyl-inositol-anchored proteins and adhesion molecules in human neutrophils. The method described here should serve as a unique additional approach for the study of topology and dynamics of molecules in biomembranes.     

A simple method for computing exact probabilities of mutation numbers

We describe a method for the recursive computation of exact probability distributions for the number of neutral mutations segregating in samples of arbitrary size and configuration. Construction of the recursions requires only characterization of evolutionary changes as a Markov process and determination of one-step transition matrices. We address the pattern of nucleotide diversity at a neutral marker locus linked to a determinant of mating type. Under a reformulation of parameters, the method also applies directly to metapopulation models with island migration among demes. Characterization of complete probability distributions facilitates parameter estimation and hypothesis testing by likelihood- as well as moment-based approaches.     

A simple and reliable method for the localization in cell culture of single identifiable cells for ultrastructural analyses

A simple method for relocating single cells in monolayer cultures for subsequent morphological or ultrastructural analysis is reported. This consists of producing, on the culture dish surface, a nontoxic carbon grid that is preserved during processing for either transmission (TEM) or scanning (SEM) electron microscopy. For TEM studies these grids are readily transferred along with the cells into the embedding plastic, and thus individual grid squares containing a cell(s) of interest can be quickly located, remounted, and sectioned. These grids may be useful for ultrastructural analyses of single cells previously studied electrophysiologically or after microinjection of macromolecules.     

Cilia regeneration in Tetrahymena : A simple reproducible method for producing large numbers of regenerating cells

We have developed an improved medium in which Tetrahymena can be deciliated by gentle shearing. The cells remain viable and regenerate a new complement of cilia. Unlike previous methods for viable deciliation of Tetrahymena, this method is easily adaptable to large numbers of cells, to cells in different stages of the life cycle (growing, starved, conjugating), and to both commonly studied species, T. thermophila and T. pyriformis. Starved T. thermophila deciliated by this method regained motility by 1 h, regenerated oral apparatus by 4.0 h and restored tubulin in cilia at a linear rate of about 3 pg h⁻¹ cell⁻¹.     

A rapid and simple method for screening large numbers of recombinant DNA clones

A simple and rapid method has been described for the isolation of plasmid, phagemid and phage DNAs. Hundreds of recombinant clones can be screened in one day employing this method. It takes half an hour to prepare plasmid DNA from ten clones, and the DNA prepared from a single colony using this method is of sufficient quality and in sufficient amount to perform at least five restriction digestions. This method eliminates the need for RNase treatment and phenol chloroform extraction if the plasmids are needed only for the restriction digestion. If needed, RNAs can be removed after restriction digestion by adding RNase and incubating for two minutes at room temperature. After RNase treatment and phenol/chloroform extraction, the plasmid DNA serves as a good template for sequencing. The DNA can be stored at -20 degrees C for over eight weeks.     

微囊藻群体细胞数量估算的一种简单方法

采用加酸水解和人工计数的方法,对巢湖铜绿微囊藻、放射微囊藻、惠氏微囊藻、坚实微囊藻、绿色微囊藻、挪氏微囊藻、水华微囊藻、鱼害微囊藻所含细胞数进行了估算.通过统计分析,建立了微囊藻群体最大投影面积与所含细胞数的回归方程模型,通过这些模型可以估算群体微囊藻所含的细胞数.