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In the present study, a high yield of isolated protoplasts from the agronomically important crop Fagopyrum esculentum was obtained by applying a mixture of cellulase, pectolyase, and driselase. We demonstrated that the yield of morphogenic callus-derived protoplasts was 1 × 106 protoplasts per g of fresh tissue. For hypocotyls used as the protoplast source, the number of released cells was twice lower. The protoplasts, embedded in an agarose matrix and cultured in a modified Kao and Michayluk media supplemented with phytosulfokine, re-enter the cell cycle and start to develop, forming microcalli. The plating efficiency was about 20% in the case of hypocotyl- and morphogenic callus-derived protoplasts. For plant regeneration, the medium was supplemented with different combinations of cytokinin. Somatic embryogenesis and organogenesis occur during the cultivation of the protoplast-derived tissues, depending on the applied protoplast source. For the first time, an effective protoplast-to-plant system for F. esculentum has been developed. Key message: Morphogenic callus- and hypocotyl-derived protoplasts of buckwheat after embedding in agarose beads and culture in phytosulfokine enriched medium regenerated into plants. [ABSTRACT FROM AUTHOR]

Background: Fagopyrum tataricum (Tartary buckwheat) is a valuable crop of great nutritional importance due to its high level of bioactive compounds. Excellent opportunities to obtain plants with the high level or the desired profile of valuable metabolites may be provided by in vitro cultures. Among known in vitro techniques, protoplast technology is an exciting tool for genetic manipulation to improve crop traits. In that context, protoplast fusion may be applied to generate hybrid cells between different species of Fagopyrum. To apply protoplast cultures to the aforementioned approaches in this research, we established the protoplast-to-plant system in Tartary buckwheat. Results: In this work, cellulase and pectinase activity enabled protoplast isolation from non-morphogenic and morphogenic callus (MC), reaching, on average, 2.3 × 106 protoplasts per g of fresh weight. However, to release protoplasts from hypocotyls, the key step was the application of driselase in the enzyme mixture. We showed that colony formation could be induced after protoplast embedding in agarose compared to the alginate matrix. Protoplasts cultured in a medium based on Kao and Michayluk supplemented with phytosulfokine (PSK) rebuilt cell walls, underwent repeated mitotic division, formed aggregates, which consequently led to callus formation. Plating efficiency, expressing the number of cell aggregate formed, in 10-day-old protoplast cultures varied from 14% for morphogenic callus to 30% for hypocotyls used as a protoplast source. However plant regeneration via somatic embryogenesis and organogenesis occurred only during the cultivation of MC-derived protoplasts. Conclusions: This study demonstrated that the applied protoplast isolation approach facilitated the recovery of viable protoplasts. Moreover, the embedding of protoplasts in an agarose matrix and supplementation of a culture medium with PSK effectively stimulated cell division and further development of Tartary buckwheat protoplast cultures along with the plant regeneration. Together, these results provide the first evidence of developing a protoplast-to-plant system from the MC of Fagopyrum tataricum used as source material. These findings suggest that Tartary buckwheat's protoplast cultures have potential implications for the species' somatic hybridization and genetic improvement. [ABSTRACT FROM AUTHOR]

The mutant distribution that accommodates both fitness and plating efficiency is an important class of the Luria–Delbrück distribution. Practical algorithms for computing this distribution do not coincide with the theoretically most elegant ones, as existing generic methods often either produce unreliable results or freeze the computational process altogether when employed to solve real-world research problems. Exploiting properties of the hypergeometric function, this paper offers an algorithm that considerably expands the scope of application of this important class of the Luria–Delbrück distribution. An integration method is also devised to complement the novel algorithm. Asymptotic properties of the mutant probability are derived to help gauge the new algorithm. An illustrative example and simulation results provide further guidelines on the use of the new algorithm. [ABSTRACT FROM AUTHOR]

Comparison of microbial mutation rates under the Luria-Delbrück protocol is a routine laboratory task. However, execution of this important task has been hampered by the lack of proper statistical methods. Visual inspection or improper use of the t test and the Mann-Whitney test can impair the quality of genetic research. This paper proposes a unified framework for constructing likelihood ratio tests that overcome three important obstacles to the proper comparison of microbial mutation rates. Specifically, algorithms for likelihood ratio tests have been devised that allow for partial plating, differential growth rates and unequal terminal cell population sizes. The new algorithms were assessed by computer simulations. In addition, a strategy for multiple comparison was illustrated by reanalyzing the experimental data from a study of bacterial resistance against tuberculosis antibiotics. [ABSTRACT FROM AUTHOR]

Protoplasts of four Daucus carota subspecies and three wild Daucus species were isolated from 2-week-old shoot cultures during overnight incubation in an enzyme mixture composed of 1 % (w/v) cellulase Onozuka R-10 and 0.1 % (w/v) pectolyase Y-23. Before the culture, they were embedded in autoclave- or filter-sterilized alginate solution. Modified thin alginate layer (TAL) and extra thin alginate film (ETAF) techniques were applied for protoplast immobilization. A rich mineral-organic medium based on the formulation of Kao and Michayluk supplemented with 0.1 mg l 2,4-dichlorophenoxyacetic acid, 0.2 mg l zeatin, and optionally 100 nM phytosulfokine (PSK), a peptidyl plant growth factor, was used for protoplast culture. Plating efficiency was genotype-dependent and in 40-day-old cultures, it varied from 10 % for Daucus pusillus to 73 % for D. carota subsp. sativus. A considerably higher ability in colony formation was observed in the modified TAL culture system using filter-sterilized alginate and in the presence of PSK in the protoplast culture medium. Plant regeneration through somatic embryogenesis stimulated by PSK application occurred for five out of the seven Daucus accessions used in the present study. We believe our data may facilitate the use of wild Daucus in somatic hybridization with cultivated carrot. [ABSTRACT FROM AUTHOR]

Poly(N-isopropyl acrylamide) (pNIPAM) is one of the most popular stimulus-responsive polymers for research. It is especially of great interest in the field of tissue engineering. While it is known that the NIPAM monomer is toxic, there is little conclusive research on the cytotoxicity of the polymer. In this work, the relative biocompatibility of the NIPAM monomer, pNIPAM, and pNIPAM-coated substrates prepared using different polymerization (free radical and plasma polymerization) and deposition (spin coating and plasma polymerization) techniques was evaluated using appropriate cytotoxicity tests (MTS, Live/Dead, plating efficiency). Four different mammalian cell types (endothelial, epithelial, smooth muscle, and fibroblasts) were used for the cytotoxicity testing. The pNIPAM-coated surfaces were evaluated for their thermoresponse and surface chemistry using X-ray photoelectron spectroscopy and goniometry. We found that while cell viability on pNIPAM surfaces decreases when compared to controls, the viability also seems to be deposition type dependent, with sol–gel based pNIPAM surfaces being the least biocompatible. Long term experiments proved that all pNIPAM-coated surfaces were not cytotoxic to the four cell types evaluated in a direct contact test. Plating efficiency experiments did not show cytotoxicity. Cellular sensitivity to pNIPAM and to the NIPAM monomer varied depending on cell type. Endothelial cells consistently showed decreased viability after 48 hours of exposure to pNIPAM extracts and were more sensitive than the other cell lines to impurities in the polymer. [ABSTRACT FROM AUTHOR]

Protoplasts isolated from three accessions of cultivated carrot and 5-day-old protoplast-derived aggregates were subjected to selection to identify somaclonal variants with enhanced tolerance to the fungal disease black rot incited by Alternaria radicina. Different concentrations [1, 2, 3.5, 5, 10, 20, 35 and 50 % (v/v)] of a fungal culture filtrate (FCF) from 2-week-old liquid cultures of A. radicina were used. Protoplasts and aggregates were subjected to short-term selection for a period of 10 days. All FCF concentrations added to the cultures on the day of isolation decreased protoplast survival frequency and plating efficiency, while FCF applied 5 days later inhibited cell divisions in 5-50 % concentrations. The responses of protoplasts to the treatment were genotype dependent. Most R0 plants were regenerated in all accessions from cell lines grown with 1 % FCF, while only a few plants were produced from 2 to 3.5 % FCF-treated cultures of 'Dolanka' and the breeding line '9304B', respectively. Nineteen-percent of putative stress-tolerant regenerants were tetraploids, while only 5 % tetraploids were observed in the control. The incidence of unique random amplified polymorphic DNA fragments indicating possible chromosomal rearrangements was low and did not differ among regenerants after selection and those derived from the control. Mobilization of miniature inverted repeat transposable elements was not observed. Some R0 individuals regenerated both from FCF-treated and untreated cultures showed lower susceptibility to A. radicina in a laboratory assay in comparison to control plants grown from seed. Regenerants from FCF-treated cultures showed lower frequency of flowering plants and a higher rate of male sterility. Pollen viability of the putative stress-tolerant regenerants varied over a wide range (6-98 %), independently of in vitro selection conditions. Our data suggest that A. radicina FCF may be feasible for the in vitro selection to generate plants with superior phenotypic performance against A. radicina. [ABSTRACT FROM AUTHOR]

The clonogenic assay is a well-established in vitro method for testing the survival and proliferative capability of cells. It can be used to determine the cytotoxic effects of various treatments including chemotherapeutics and ionizing radiation. However, this approach can also characterize cells with different phenotypes and biological properties, such as stem cells or cancer stem cells. In this study, we implemented a faster and more precise method for assessing the cloning efficiency of cancer stem-like cells that were characterized and separated using a high-speed cell sorter. Cell plating onto a microplate using an automatic cell deposition unit was performed in a single-cell or dilution rank mode by the fluorescence-activated cell sorting method. We tested the new automatic cell-cloning assay (ACCA) on selected cancer cell lines and compared it with the manual approach. The obtained results were also compared with the results of the limiting dilution assay for different cell lines. We applied the ACCA to analyze the cloning capacity of different subpopulations of prostate and colon cancer cells based on the expression of the characteristic markers of stem (CD44 and CD133) and cancer stem cells (TROP-2, CD49f, and CD44). Our results revealed that the novel ACCA is a straightforward approach for determining the clonogenic capacity of cancer stem-like cells identified in both cell lines and patient samples. © 2013 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

Understanding the behavior of date palm (Phoenix dactylifera L.) cell suspension growth and differentiation would foster effective utilization for mass micropropagation and various in vitro investigations. The objectives of this study were to define the growth curve, identify the optimum plating density, and examine the efficiency of somatic embryogenesis of date palm suspension culture on solid and liquid media. Cell suspensions were established from shoot tip-induced callus of cv. Barhee inoculated in MS medium containing 10 mg l-1 naphthaleneacetic acid and 1.5 mg l-1 2-isopentenyladenine. Various growth phases including lag, exponential, linear, progressive deceleration, and stationary, along with their specific onsets and durations, were identified based on packed cell volume method. The growth pattern characterizing the exponential phase in date palm cell suspensions commenced 4 weeks after culture initiation which makes this period the most suitable for subculturing, assessment of the effects tissue culture factors, and in vitro selection. The effect of cell plating density on cell growth after transferring to solidified medium was also determined. The highest plating efficiency, 14.6%, was obtained at cell density of 10,000 cells ml-1. To stimulate somatic embryogenesis, cell suspension masses were transferred to agar or liquid media devoid of phytohormones. Plant regeneration was marked by the development of globular somatic embryos which progressively matured and germinated. Culturing suspension mass in liquid medium expedited regeneration and resulted in 3.5-fold more somatic embryos than agar medium. [ABSTRACT FROM AUTHOR]

An easy and effective regeneration system from leaf- and hypocotyl-derived protoplasts was established for carrot. The protoplast isolation efficiency after preplasmolytic treatment and digestion of source material in enzyme mixture consisted of 1% cellulase Onozuka R-10 and 0.1% pectolyase Y-23 reached on average 3 × 10 and 10 protoplasts per g of leaf and hypocotyl tissue, respectively. A modified thin alginate layer technique was applied for the protoplast culture. Direct somatic embryogenesis on a simplified Kao and Michayluk medium in the presence of 2,4-D and zeatin occurred during cultivation of both leaf- and hypocotyl-derived protoplasts for all accessions used. Morphologically normal plants were produced at very high efficiency within two months after initiation of the protoplast culture. Ninety three percent of in vitro derived plants were diploids. Pollen viability and seed set after self-pollination were similar to those of plants obtained from seeds. [ABSTRACT FROM AUTHOR]

Scaevola, characterised by unique fan-shaped flowers, is an Australian endemic ornamental having a great commercial potential. The breeding of Scaevola however is limited due to poor seed germination, therefore it is critical to understand the embryogenesis in Scaevola so as to facilitate its breeding improvement programs. Direct differentiation of embryo structures was first reported here in mesophyll protoplast cultures of Scaevola aemula. The isolated protoplasts initiated cell division when cultured in KM or MS medium. Higher plating efficiencies were observed in the medium containing a combination of NAA and BAP in contrast to 2,4-D and BAP. The formation of globular embryo structures was successfully achieved. This protoplast culture system can be utilized as an experimental platform for the study of embryogenic differentiation of cells. It may open new vistas to investigate the seed development at molecular and cellular levels in Scaevola and related Australian native plants that are well known for their low seed viability and germination. [ABSTRACT FROM AUTHOR]

The extreme sensitivity of many Archaea to oxygen is a major obstacle for their cultivation in the laboratory and the development of archaeal genetic exchange systems. The technique of Balch and Wolfe (1976) is suitable for the cultivation of anaerobic Archaea but involves time-consuming procedures such as the use of air locks and glove boxes. We describe here a procedure for the cultivation of anaerobic Archaea that is more convenient and faster and allows the preparation of liquid media without the use of an anaerobic chamber. When the reducing agent sodium sulfide (Na2S) was replaced by sodium sulfite (Na2SO3), anaerobic media could be prepared without protection from oxygen outside an anaerobic chamber. Exchange of the headspace of serum bottles by appropriate gases was sufficient to maintain anaerobic conditions in the culture media. Organisms that were unable to utilize sulfite as a source for cellular sulfur were supplemented with hydrogen sulfide. H2S was simply added to the headspace of serum bottles by a syringe. The use of H2S as a source for sulfur minimized the precipitation of cations by sulfide. Representatives of 12 genera of anaerobic Archaea studied here were able to grow in media prepared by this procedure. For the extremely oxygen-sensitive organism Methanococcus thermolithotrophicus, we show that plates could be prepared outside an anaerobic chamber when sulfite was used as reducing agent. The application of this method may faciliate the cultivation and handling of extreme anaerobic Archaea considerably. [ABSTRACT FROM AUTHOR]

The influence of the exogenous polyamines: putrescine, spermidine and spermine, on the frequency of protoplast divisions for 2 genotypes of sugar beet (Beta vulgaris L.) was analyzed. Protoplasts were cultured by the agarose disk method on Saunders and Doley medium supplemented with either hormones or polyamines, or hormones combined with polyamines. The latter supplement led to a statistically significant increase in plating efficiency. The improvement in division index was caused mainly by spermine. [ABSTRACT FROM AUTHOR]

The requirements for establishment and survival of primary cultures of larval amphibian liver cells were investigated. Plating efficiency was found to be enhanced by a collagen substrate, by diluted conditioned medium from an adult Xenopus kidney cell line and by high initial cell densities. Plating efficiency was highest at a tonicity of 165-220 mOsm/kg. In cultures with undiluted conditioned medium the increase in cell number was 50-60% greater than in controls, where it was about 2-fold between day 3 and 6 of culture. Conditioned medium from the Xenopus kidney cell line is assumed to contain at least two components, which are effective at different concentrations and stimulate either plating efficiency and cell aggregation or cell proliferation. In cultures without collagen sheets, cell flattening is greatly reduced, indicating that cell shape is also dependent upon the substrate. [ABSTRACT FROM AUTHOR]

Suspensions of Haplopappus gracilis cells, containing about 80% free cells, were obt ained from log-phase cultures by filtration through 3 nylon sieves having decreasing mesh widths from 297, 210 and 88 μm. From the free cell suspensions, 75 to 90% of the cells developed into visible colonies when the plating procedure was divided into two steps: a) plating the cells at high concentration in soft agar on feeder agar; b) replating the resulting aggregations at appropriate concentrations on fresh feeder agar. From the results, it is inferred that, in the replating step, the volume of the inoculum is the deciding factor which influences the resulting plating efficiency. [ABSTRACT FROM AUTHOR]

The growth-promoting effects of nurse cells of carrot, tomato, potato, maize, bean, carnation and two species of tobacco were studied on carrot, tomato, tobacco and potato cells plated at low densities. In an area immediately below the nurse cells the plating efficiency was very high and found to be independent of cell density. In an area outside the nurse cells, in some cases, the plating efficiency tended to be much higher in combinations with cells from a heterologous source as compared with those from a homologous source. Moreover, in the same area with some combinations the plating efficiency decreased when cell density was lowered, while with other combinations this phenomenon did not occur. This decrease was independent of the absolute value of plating efficiency. In experiments in which the concentration of conditioning factors was presumably changed, no significant difference in the plating efficiency was noticed. We therefore suggest that different plating efficiencies observed with heterologous nurse cells were not due to a higher level of conditioning factors, but rather to the production of different types of conditioning factors that are presumably degraded with different efficiency. [ABSTRACT FROM AUTHOR]

High yields (2.3 × 105 to 1.3 × 106 protoplasts/g.f.wt.) of isolated protoplasts were obtained from cotyledons of Citrus sinensis (L.) Osb. `Valencia', Osmotic potential of the medium and enzyme concentrations were important in obtaining high viability of preparations as indicated by FDA fluorescence. Adding malt extract to a Murashige-Tucker basal medium increased plating efficiencies somewhat, but not the rate or duration of cell division. However, modifying the NAA and kinetin concentration optimized plating efficiencies (up to 20%) of protoplasts and also the rate or duration of cell division. The highest plating efficiency and number of cells per colony were obtained on a defined medium containing NAA (15 μ M) and kinetin (4.6 μ M). Coincidence of percentage protoplast viability after 13 days (assessed by FDA fluorescence) with plating efficiency after 21 days indicates that FDA fluorescence is an accurate indicator of citrus protoplast viability. [ABSTRACT FROM AUTHOR]

Using skin fibroblastlike cells from 10 healthy volunteers, 12 to 54 yr old, we measured, in replicates, seeding efficiency (SE), plating efficiency (PE), and population doublings (PD) during 2 wk of growth and the cumulative replicative life span (CRLS). We determined the effects of donor age and of in vitro cell age on the test results and test precision. We found that (a) On the average, SE was 82%±24 SD, PE was 12%±9 SD, PD was 7.3 doublings ±2.5 SD, and CRLS was 31.1 doublings ±9.6 SD. (b) Plating efficiency and PD correlated well with each other and with CRLS whereas SE did not correlate with PE, PD, or CRLS. (c) Plating efficiency and PD were affected negatively by increasing passage levels and by donor ages, but the influence of age was evident only for cells from early passages. Seeding efficiency was not affected by either of the two variables. (d) Variations in PD from early passages explained half of the donor-to-donor variation in CRLS whereas PD from later passages had little or no predictive power. (e) Of the two short term tests, PD had better precision than PE. For replicate runs with cells from early passages the coefficient of variation was 44% for PE and 15% for PD. The results show that PD for cells from early passages in mass culture give rapid results, which express the in vitro replicating potential of cells from different donors with greater precision than do measurements of CRLS. [ABSTRACT FROM AUTHOR]

The toxic effects of 6-hydroxydopamine on the human neuroblastoma cell line SK-N-SH-SY5Y (SY5Y) and the Chinese hamster ovary (CHO) cell line were measured with five viability assays. Four of the assays (attachment efficiency, plating efficiency, amino acid incorporation into acid-precipitable proteins, and Trypan Blue dye exclusion) showed higher drug susceptibility in SY5Y cells than CHO cells. Only growth inhibition (proliferation index) gave results indicating greater sensitivity in CHO cells. Over a time span of 48 hr, injured cell populations lost vital functions in the following order: attachment ability, amino acid incorporation, proliferative capacity, and dye exclusion. Recovery of each of the functions occurred in sublethally injured populations. Monitoring the extinction and recovery of vital functions permitted the accurate determination of a drug concentration (30 μg/ml) selectively toxic for SY5Y cells. A strong correlation was noted between relative values for amino acid incorporation 3 hr after drug treatment, attachment efficiency at 24 hr, and dye exclusion at 24 and 48 hr. We concluded that Trypan Blue dye exclusion and amino acid incorporation were suitable methods for comparing the effects of cytotoxins on different cell lines, provided they were performed at the appropriate time after treatment with the toxin. The combined techniques yield both population and individual cell data, are simple to do, and are applicable to nondividing cell populations. [ABSTRACT FROM AUTHOR]

Electric field pulses, ranging from 250 to 2000 V and of 10 to 50 μsec duration, were assessed for their effect on the growth in culture of isolated protoplasts of Glycine canescens, Prunus avium × pseudocerasus, Pyrus communis, Solanum dulcamara and Solanum viarum. Three successive voltage pulses between 250 and 1000 V caused a small decrease in protoplast viability, but promoted cell division and enhanced significantly the plating efficiency. A higher percentage of electro-pulsed protoplasts showed sustained growth in culture to the microcallus stage compared to untreated protoplasts. The rate of cell division was also stimulated in electro-treated protoplasts. These observations are discussed in relation to present knowledge of the effects of electrical treatments on plant and animal cells. [ABSTRACT FROM AUTHOR]

It has been shown previously that the initial product of mushroom tyrosinase-catalysed oxidation of the monophenol 4-hydroxyanisole (4HA) is 4-methoxy ortho benzoquinone (4-MOB). This study presents evidence that 4-MOB is primarily responsible for the cytotoxicity of 4HA oxidation products in vitro. Equivalent toxicity in a model system was produced by products of tyrosinase catalysed oxidation of 4HA and by synthetic 4-MOB. Cytotoxicity was estimated both by a blebbing assay and by plating efficiency of exposed cells. HPLC analysis of the reaction mixture revealed a positive correlation between cytotoxicity and 4-MOB concentration. [ABSTRACT FROM AUTHOR]

Mononuclear cells, harvested from fresh human bone marrow specimens by density gradient separation, were suspended in phosphate buffered saline and analyzed by flow cytometry in terms of the forward and right angle scattering of the incident light. The rectilinear distribution, obtained by plotting the intensity of light scattered in the forward and right angle directions, contained three regions of interest in which the percentage of cells (Mean ± standard deviation) with respect to the total was as follows: Region 1: 17.6±9.9; region 2: 5.3±1.4; region 3: 71.7±9.4. Cells from each region were sorted by flow cytometry and plated in semi-solid agar containing cell conditioned medium supportive of myeloid colony formation. Cells from region 2 contained the majority of progenitor cells that gave rise to such colonies at a plating efficiency that rose in proportion to the extent by which the region 2 cells in samples was increased through sorting. This increase in plating efficiency was 6 to 43 fold. Thus, region 2 of the cytometric distribution of cells from normal, unstained human bone marrows was a good source of myeloid progenitor cells. [ABSTRACT FROM AUTHOR]

Fetal bovine sera from each of three different commercial sources were tested for their ability to support cloning of human fibroblastoid cells in vitro. Cloning efficiencies varied according to serum source. Serum (10 samples) from company A did not support growth, while sera (10 samples) from companies B and C provided adequate to excellent conditions for cloning and growth. Cells from neonatal foreskin or embryonic lung responded to each serum similarly. Bovine serum albumin type H7 from company C supported cell growth in media without serum. Sera containing 1.0 ng per ml or more of progesterone inhibited growth, whereas sera containing less than 1.0 ng per ml supported cloning and growth. In the low progesterone sera, the concentration of 17-β-estradiol exceeded 100 pg per ml. Growth supporting sera could be made non-supportive by adding 0.1 μg per ml of progesterone. The addition to non-supportive sera of 0.1 μg per ml of 17-β-estradiol or hydrocortisone made these sera supportive of cell growth. Addition of estrogen or hydrocortisone to a culture medium that inhibits growth, with subsequent reversal of the inhibitory effect, implies that these hormones competitively regulate growth of responsive cells in vitro. [ABSTRACT FROM AUTHOR]

Mild sonication was used to obtain single cell suspensions of Paracoccidioides brasiliensis. These cells were intact by microscopic criteria. Direct cell counts in a given inoculum and colony formation on various media were used to determine plating efficiency. Sonicated and nonsonicated cell suspensions were used to study plating efficiency and to estimate viability by means of vital dyes. Methylene blue, Erythrosin B, and Janus green were unreliable when used with P. brasiliensis, but vital dyes were accurate when tested with Candida albicans. Acridine orange gave more meaningful results of viability. Estimates of viability, however, changed significantly as a result of relatively minor alterations in the composition of the suspending medium. In initial experiments, the plating efficiency of P. brasiliensis was dismally low. It descended abruptly with increasing dilution of inoculum. Efficiency was much improved if horse serum was added to brain heart infusion plates or if glucose glycine yeast extract (GGY) plates were incubated at room temperature and mycelial colonies were counted. With the technique we report, current plating efficiency of sonicated suspensions is of the order of 25 %. Our results and procedures have an important bearing upon those studies concerned with in vitro killing of P. brasiliensis in suspensions or with isolating this fungus from clinical or environmental specimens. [ABSTRACT FROM AUTHOR]

Background: The clonogenic assay is a versatile and frequently used tool to quantify reproductive cell survival in vitro. Current state-of-the-art analysis relies on plating efficiency-based calculations which assume a linear correlation between the number of cells seeded and the number of colonies counted. The present study was designed to test the validity of this assumption and to evaluate the robustness of clonogenic survival results obtained.Methods: A panel of 50 established cancer cell lines was used for comprehensive evaluation of the clonogenic assay procedure and data analysis. We assessed the performance of plating efficiency-based calculations and examined the influence of critical experimental parameters, such as cell density seeded, assay volume, incubation time, as well as the cell line-intrinsic factor of cellular cooperation by auto-/paracrine stimulation. Our findings were integrated into a novel mathematical approach for the analysis of clonogenic survival data.Results: For various cell lines, clonogenic growth behavior failed to be adequately described by a constant plating efficiency, since the density of cells seeded severely influenced the extent and the dynamics of clonogenic growth. This strongly impaired the robustness of survival calculations obtained by the current state-of-the-art method using plating efficiency-based normalization. A novel mathematical approach utilizing power regression and interpolation of matched colony numbers at different irradiation doses applied to the same dataset substantially reduced the impact of cell density on survival results. Cellular cooperation was observed to be responsible for the non-linear clonogenic growth behavior of a relevant number of cell lines and the impairment of survival calculations. With 28/50 cell lines of different tumor entities showing moderate to high degrees of cellular cooperation, this phenomenon was found to be unexpectedly common.Conclusions: Our study reveals that plating efficiency-based analysis of clonogenic survival data is profoundly compromised by cellular cooperation resulting in strongly underestimated assay-intrinsic errors in a relevant proportion of established cancer cell lines. This severely questions the use of plating efficiency-based calculations in studies aiming to achieve more than semiquantitative results. The novel approach presented here accounts for the phenomenon of cellular cooperation and allows the extraction of clonogenic survival results with clearly improved robustness. [ABSTRACT FROM AUTHOR]

Radiation induced stimulation of plating efficiency of free plant cells was observed following irradiation with X-rays (1.25 Gy, dose rate 3.1 Gy.nin) and fission neutrons (1.5 Gy, dose rate 0.05 Gy.nin). The dose range where the radiation stimulation effect is manifest is inversely correlated with the applied dose rate. The results are discussed in view of the radiation induced stimulation as it is applied in agricultural practice. [ABSTRACT FROM AUTHOR]