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Cytometry. Part a : the Journal of the... Sep 2006One of the major factors which influences the chromosome purity achievable particularly during high speed sorting is the analytical resolution of individual chromosome...
BACKGROUND
One of the major factors which influences the chromosome purity achievable particularly during high speed sorting is the analytical resolution of individual chromosome peaks in the flow karyotype, as well as the amount of debris and fragmented chromosomes. We have investigated the factors involved in the preparation of chromosome suspensions that influence karyotype resolution.
METHODS
Chromosomes were isolated from various human and animal cell types using a series of polyamine buffer isolation protocols modified with respect to pH, salt concentration, and chromosome staining time. Each preparation was analyzed on a MoFlo sorter (DAKO) configured for high speed sorting and the resolution of the flow karyotypes compared.
RESULTS
High resolution flow cytometric data was obtained with chromosomes optimally isolated using hypotonic solution buffered at pH 8.0 and polyamine isolation buffer (with NaCl excluded) between pH 7.50 and 8.0. Extending staining time to more than 8 h with chromosome suspensions isolated from cell lines subjected to sufficient metaphase arrest times gave the best result with the lowest percentage of debris generated, tighter chromosome peaks with overall lower coefficients of variation, and a 1- to 5-fold increase in the yield of isolated chromosomes.
CONCLUSIONS
Optimization of buffer pH and the length of staining improved karyotype resolution particularly for larger chromosomes and reduced the presence of chromosome fragments (debris). However, the most interesting and surprising finding was that the exclusion of NaCl in PAB buffer improved the yield and resolution of larger chromosomes.
Topics: Animals; Buffers; Cell Line; Chromosomes; Humans; Hydrogen-Ion Concentration; Karyotyping; Male; Staining and Labeling
PubMed: 16969800
DOI: 10.1002/cyto.a.20330 -
Biophysical Journal Nov 2016We consider the stationary solution for the Ca concentration near a point Ca source describing a single-channel Ca nanodomain, in the presence of a single mobile buffer...
We consider the stationary solution for the Ca concentration near a point Ca source describing a single-channel Ca nanodomain, in the presence of a single mobile buffer with one-to-one Ca binding stoichiometry. Previously, a number of Ca nanodomains approximations have been developed, for instance the excess buffer approximation (EBA), the rapid buffering approximation (RBA), and the linear approximation (LIN), each valid for appropriate buffering conditions. Apart from providing a simple method of estimating Ca and buffer concentrations without resorting to computationally expensive numerical solution of reaction-diffusion equations, such approximations proved useful in revealing the dependence of nanodomain Ca distribution on crucial parameters such as buffer mobility and its Ca binding properties. In this study, we present a different form of analytic approximation, which is based on matching the short-range Taylor series of the nanodomain concentration with the long-range asymptotic series expressed in inverse powers of distance from channel location. Namely, we use a "dual" Padé rational function approximation to simultaneously match terms in the short- and the long-range series, and we show that this provides an accurate approximation to the nanodomain Ca and buffer concentrations. We compare this approximation with the previously obtained approximations and show that it yields a better estimate of the free buffer concentration for a wide range of buffering conditions. The drawback of our method is that it has a complex algebraic form for any order higher than the lowest bilinear order, and cannot be readily extended to multiple Ca channels. However, it may be possible to extend the Padé method to estimate Ca nanodomains in the presence of cooperative Ca buffers with two Ca binding sites, the case that existing methods do not address.
Topics: Binding Sites; Buffers; Calcium; Calcium Channels; Calcium Signaling; Cell Membrane; EF Hand Motifs; Models, Biological
PubMed: 27806286
DOI: 10.1016/j.bpj.2016.09.019 -
Contraception Apr 2016Reported vaginal and seminal fluid simulants have complex compositions with multiple preparatory steps that contribute to physical instability. We report the design and...
BACKGROUND
Reported vaginal and seminal fluid simulants have complex compositions with multiple preparatory steps that contribute to physical instability. We report the design and characterization of stable and simplified buffers that mimic the salient physical/chemical properties of the physiological fluids.
STUDY DESIGN/METHODS
Human cervicovaginal and seminal fluid samples were collected and buffering capacity was determined. The major buffering species were identified from published compositions of reproductive tract fluids. These values were used to compute the composition of vaginal and seminal fluid simulants. Ionic strength, buffering capacities, pH and osmolalities were then calculated or experimentally determined. Finally, cytotoxicity was evaluated in HEC-1-A cells and 3D reconstructed EpiVaginal™ tissue (VEC-100-FT) using naïve cells/tissue and nonoxynol-9 as controls.
RESULTS
The use of calculated amounts of conjugate acid and base for buffer development resulted in compositions that did not require endpoint pH adjustment and could be formulated as stable 10× concentrates. Furthermore, due to the absence of complex divalent salts, all our proposed simulants were stable at 4 °C for 1 month whereas precipitation and pH and osmolality changes were noted in reported buffers. Experimental determination of buffering capacities yielded similar values for undiluted cervicovaginal fluid (β4.2-5.2=35.6 ± 12.3 mM, N=7) and human seminal fluid (β7-6=37.5 ± 5 mM, N=3). All neat simulants showed significant cytotoxicity in HEC-1-A cells but were well tolerated by organotypic vaginal tissue.
CONCLUSIONS
We report revised and improved compositions of buffers mimicking salient properties of vaginal and seminal fluid necessary for in vitro product evaluation.
IMPLICATIONS
To support research in reproductive health and in particular drug delivery, we have designed and characterized stable new media to mimic these important fluids that can be used in a variety of in vitro studies.
Topics: Bioengineering; Body Fluids; Buffers; Chemical Phenomena; Chemical Precipitation; Drug Delivery Systems; Female; Humans; Hydrogen-Ion Concentration; Male; Osmolar Concentration; Semen; Vagina
PubMed: 26585883
DOI: 10.1016/j.contraception.2015.11.008 -
Cell Systems Nov 2017Buffering, the use of reservoirs of molecules to maintain concentrations of key molecular species, and negative feedback are the primary known mechanisms for robust...
Buffering, the use of reservoirs of molecules to maintain concentrations of key molecular species, and negative feedback are the primary known mechanisms for robust homeostatic regulation. To our knowledge, however, the fundamental principles behind their combined effect have not been elucidated. Here, we study the interplay between buffering and negative feedback in the context of cellular homeostasis. We show that negative feedback counteracts slow-changing disturbances, whereas buffering counteracts fast-changing disturbances. Furthermore, feedback and buffering have limitations that create trade-offs for regulation: instability in the case of feedback and molecular noise in the case of buffering. However, because buffering stabilizes feedback and feedback attenuates noise from slower-acting buffering, their combined effect on homeostasis can be synergistic. These effects can be explained within a traditional control theory framework and are consistent with experimental observations of both ATP homeostasis and pH regulation in vivo. These principles are critical for studying robustness and homeostasis in biology and biotechnology.
Topics: Adenosine Triphosphate; Animals; Buffers; Feedback; Homeostasis; Hydrogen-Ion Concentration; Mammals; Models, Biological
PubMed: 29055671
DOI: 10.1016/j.cels.2017.09.013 -
Applied Microbiology Apr 1970A previously described agar-diffusion technique for microbioassay of antimicrobial agents has been modified to increase sensitivity of the technique and to extend the...
A previously described agar-diffusion technique for microbioassay of antimicrobial agents has been modified to increase sensitivity of the technique and to extend the range of antimicrobial agents to which it is applicable. This microtechnique requires only 0.02 ml of an unknown test sample for assay, and is capable of measuring minute concentrations of antibiotics in buffer, serum, and urine. In some cases, up to a 20-fold increase in sensitivity is gained relative to other published standardized methods and the error of this method is less than +/-5%. Buffer standard curves have been established for this technique, concurrently with serum standard curves, yielding information on antimicrobial serum-binding and demonstrating linearity of the data points compared to the estimated regression line for the microconcentration ranges covered by this technique. This microassay technique is particularly well suited for pediatric research and for other investigations where sample volumes are small and quantitative accuracy is desired. Dilution of clinical samples to attain concentrations falling with the range of this assay makes the technique readily adaptable and suitable for general clinical pharmacological studies. The microassay technique has been standardized in buffer solutions and in normal human serum pools for the following antimicrobials: ampicillin, methicillin, penicillin G, oxacillin, cloxacillin, dicloxacillin, cephaloglycin, cephalexin, cephaloridine, cephalothin, erythromycin, rifamycin amino methyl piperazine, kanamycin, neomycin, streptomycin, colistin, polymyxin B, doxycycline, minocycline, oxytetracycline, tetracycline, and chloramphenicol.
Topics: Agar; Anti-Bacterial Agents; Bacillus cereus; Biological Assay; Bordetella; Buffers; Colorimetry; Diffusion; Humans; Methods; Sarcina; Staphylococcus; Statistics as Topic
PubMed: 4986725
DOI: 10.1128/am.19.4.573-579.1970 -
Acta Crystallographica. Section F,... Oct 2015There is strong evidence to suggest that a protein sample needs to be well folded and uniform in order to form protein crystals, and it is accepted knowledge that the...
There is strong evidence to suggest that a protein sample needs to be well folded and uniform in order to form protein crystals, and it is accepted knowledge that the formulation can have profound effects on the behaviour of the protein sample. The technique of differential scanning fluorimetry (DSF) is a very accessible method to determine protein stability as a function of the formulation chemistry and the temperature. A diverse set of 252 soluble protein samples was subjected to a standard formulation-screening protocol using DSF. Automated analysis of the DSF results suggest that in over 35% of cases buffer screening significantly increases the stability of the protein sample. Of the 28 standard formulations tested, three stood out as being statistically better than the others: these included a formulation containing the buffer citrate, long known to be `protein friendly'; bis-tris and ADA were also identified as being very useful buffers in protein formulations.
Topics: Animals; Buffers; Carbonic Anhydrases; Cattle; Fluorometry; Hydrogen-Ion Concentration; Isoelectric Point
PubMed: 26457531
DOI: 10.1107/S2053230X15012662 -
European Journal of Biochemistry Dec 1987Sepia cartilage collagen (pepsin-extracted) in acetate buffer (pH = 2.98) forms micelles at a particular concentration below which they do not normally form. The...
Physico-chemical studies of micelle formation on sepia cartilage collagen solutions in acetate buffer and its interaction with ionic and nonionic micelles. Hydrodynamic and thermodynamic studies.
Sepia cartilage collagen (pepsin-extracted) in acetate buffer (pH = 2.98) forms micelles at a particular concentration below which they do not normally form. The critical micelle concentration (cmc) of the collagen was determined in buffer as well as in SDS, cetyltrimethylammonium bromide (CTAB) and Tween-80 micellar environments at different temperatures. Mutual interaction of collagen micelles with the ionic and nonionic micelles through the formation of the mixed micelle concept has also been found. The cmc of collagen decreased in the presence of SDS and Tween-80 micelles whereas it increased in the presence of CTAB micelles. This clearly suggests that the micelle formation of collagen is facilitated by the presence of SDS and Tween-80 and hindered by CTAB micelles. The various thermodynamic parameters were estimated from viscosity measurements and the transfer of collagen into the micelles of various surfactants and the reverse phenomenon was analyzed. This analysis has also been modelled conceptually as a different phase and the results have supported the above phenomenon. Our thermodynamic results are also able to predict the exact denaturation temperature as well as the structural order of water in the collagen in various environments. The hydrated volumes, Vh, of collagen in the above environments and intrinsic viscosity were also calculated. The low intrinsic viscosity, [eta], of collagen in an SDS environment compared to buffer and other surfactant environments suggested more workable systems in cosmetic and dermatological skin care preparations. The one and two-hydrogen-bonded models of this collagen in various environments have been analyzed. The calculated thermodynamic parameters varied with the concentration of collagen. The change of thermodynamic parameters from coil-coil to random-coil conformation upon denaturation of collagen were calculated from the amount of proline and hydroxyproline residues and compared with viscometric results. Thermodynamic results suggest that the stability of the collagen in the additive environments is in the following order: SDS greater than Tween-80 greater than buffer greater than CTAB.
Topics: Acetates; Acetic Acid; Algorithms; Animals; Buffers; Cartilage; Chemical Phenomena; Chemistry, Physical; Collagen; Colloids; Micelles; Solutions; Thermodynamics; Water
PubMed: 3691510
DOI: 10.1111/j.1432-1033.1987.tb13653.x -
Microbial Cell Factories Sep 2012Buffering to achieve pH control is crucial for successful trichloroethene (TCE) anaerobic bioremediation. Bicarbonate (HCO3-) is the natural buffer in groundwater and...
BACKGROUND
Buffering to achieve pH control is crucial for successful trichloroethene (TCE) anaerobic bioremediation. Bicarbonate (HCO3-) is the natural buffer in groundwater and the buffer of choice in the laboratory and at contaminated sites undergoing biological treatment with organohalide respiring microorganisms. However, HCO3- also serves as the electron acceptor for hydrogenotrophic methanogens and hydrogenotrophic homoacetogens, two microbial groups competing with organohalide respirers for hydrogen (H2). We studied the effect of HCO3- as a buffering agent and the effect of HCO3--consuming reactions in a range of concentrations (2.5-30 mM) with an initial pH of 7.5 in H2-fed TCE reductively dechlorinating communities containing Dehalococcoides, hydrogenotrophic methanogens, and hydrogenotrophic homoacetogens.
RESULTS
Rate differences in TCE dechlorination were observed as a result of added varying HCO3- concentrations due to H2-fed electrons channeled towards methanogenesis and homoacetogenesis and pH increases (up to 8.7) from biological HCO3- consumption. Significantly faster dechlorination rates were noted at all HCO3- concentrations tested when the pH buffering was improved by providing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) as an additional buffer. Electron balances and quantitative PCR revealed that methanogenesis was the main electron sink when the initial HCO3- concentrations were 2.5 and 5 mM, while homoacetogenesis was the dominant process and sink when 10 and 30 mM HCO3- were provided initially.
CONCLUSIONS
Our study reveals that HCO3- is an important variable for bioremediation of chloroethenes as it has a prominent role as an electron acceptor for methanogenesis and homoacetogenesis. It also illustrates the changes in rates and extent of reductive dechlorination resulting from the combined effect of electron donor competition stimulated by HCO3- and the changes in pH exerted by methanogens and homoacetogens.
Topics: Bicarbonates; Buffers; Chloroflexi; Electrons; HEPES; Halogenation; Hydrogen; Hydrogen-Ion Concentration; Oxidation-Reduction; Trichloroethylene
PubMed: 22974059
DOI: 10.1186/1475-2859-11-128 -
The Journal of Biological Chemistry Feb 2022Complex diseases such as cancer and diabetes are underpinned by changes in metabolism, specifically by which and how nutrients are catabolized. Substrate utilization can...
Complex diseases such as cancer and diabetes are underpinned by changes in metabolism, specifically by which and how nutrients are catabolized. Substrate utilization can be directly examined by measuring a metabolic endpoint rather than an intermediate (such as a metabolite in the tricarboxylic acid cycle). For instance, oxidation of specific substrates can be measured in vitro by incubation of live cultures with substrates containing radiolabeled carbon and measuring radiolabeled carbon dioxide. To increase throughput, we previously developed a miniaturized platform to measure substrate oxidation of both adherent and suspension cells using multiwell plates rather than flasks. This enabled multiple conditions to be examined simultaneously, ideal for drug screens and mechanistic studies. However, like many metabolic assays, this was not compatible with bicarbonate-buffered media, which is susceptible to alkalinization upon exposure to gas containing little carbon dioxide such as air. While other buffers such as HEPES can overcome this problem, bicarbonate has additional biological roles as a metabolic substrate and in modulating hormone signaling. Here, we create a bicarbonate-buffered well-plate platform to measure substrate oxidation. This was achieved by introducing a sealed environment within each well that was equilibrated with carbon dioxide, enabling bicarbonate buffering. As proof of principle, we assessed metabolic flux in cultured adipocytes, demonstrating that bicarbonate-buffered medium increased lipogenesis, glucose oxidation, and sensitivity to insulin in comparison to HEPES-buffered medium. This convenient and high-throughput method facilitates the study and screening of metabolic activity under more physiological conditions to aid biomedical research.
Topics: Bicarbonates; Buffers; Carbon Dioxide; Cell Culture Techniques; Culture Media; HEPES; Oxidation-Reduction
PubMed: 34971704
DOI: 10.1016/j.jbc.2021.101547 -
Journal of Nuclear Medicine Technology Mar 2013The objective of this research is to determine whether there are significant differences in the (18)F-FDG produced by either the phosphate or the citrate buffer... (Comparative Study)
Comparative Study
UNLABELLED
The objective of this research is to determine whether there are significant differences in the (18)F-FDG produced by either the phosphate or the citrate buffer cassettes in the FASTlab synthesizer.
METHODS
Forty batches of (18)F-FDG were produced with each cassette and analyzed retrospectively. The analysis consisted of determining the mean radiochemical yield (RCY)-uncorrected and corrected for decay-radiochemical purity (RCP), pH, and residual solvent content (ethanol and acetonitrile). An independent t test (alpha error [α], 0.05) was performed to determine whether the differences were statistically significant.
RESULTS
The mean decay-corrected RCYs for (18)F-FDG produced by phosphate and citrate cassettes were 82.9% ± 17.4% and 79.2% ± 5.0%, respectively. The uncorrected RCY was 57.5% ± 16.7% for phosphate- and 58.8% ± 6.0% for citrate-buffered (18)F-FDG, leading to a difference of 4.4% and P value of 0.11 for corrected RCY and a difference of 2.2% and P value of 0.32 for uncorrected RCY. Thus, the RCY differences are neither statistically nor clinically significant. The mean RCPs were 99.4% ± 0.2% for the phosphate-buffered (18)F-FDG and 99.0% ± 1.1% for the citrate-buffered (18)F-FDG. There was a 0.5% difference and a P value of 0.021, meaning that the difference was statistically significant. The average pHs for (18)F-FDG produced by phosphate and citrate buffer cassettes were 5.9 ± 0.1 and 5.3 ± 0.2, respectively, resulting in a 9.6% difference and a P value close to zero (2.6 × 10(-19))-a statistically significant difference. The difference between ethanol content was also dramatic. Phosphate-buffered (18)F-FDG contained 0.08% ± 0.02% ethanol, whereas the citrate-buffered (18)F-FDG contained 0.20% ± 0.07%. No difference was found in the acetonitrile content of the 2 cassettes.
CONCLUSION
The differences in yield between cassettes are due to statistical variability. The results confirm our hypothesis that there is no significant difference in RCY. The differences seen in the statistically significant data (those with a P value > 0.05) turn out to be insignificant in a real-world setting because all values fell within the limits set by the United States Pharmacopeia and Food and Drug Administration. Therefore, determining which cassette to use is a matter of the preference of the institution.
Topics: Buffers; Citrates; Fluorodeoxyglucose F18; Hydrogen-Ion Concentration; Phosphates; Radiochemistry; Solvents
PubMed: 23318199
DOI: 10.2967/jnmt.112.112649