scholarly journals DEOXYRIBONUCLEIC ACID CYTOPHOTOMETRY OF STAINED HUMAN LEUKOCYTES III. THERMAL DENATURATION OF CHROMATIN

1974 ◽  
Vol 22 (2) ◽  
pp. 120-126 ◽  
Author(s):  
STEPHEN C. DRESKIN ◽  
BRIAN H. MAYALL
Keyword(s):  
Thermal Denaturation ◽  
Deoxyribonucleic Acid ◽  
Single Cells ◽  
Human Lymphocytes ◽  
Freeze Substitution ◽  
Enzymatic Digestion ◽  
Single Strand ◽  
Different Temperatures ◽  
Potential Methods

Thermal denaturation of chromatin has been monitored in situ in single cells using absorption cytophotometry on gallocyanin-chrome alum (GCA)-stained preparations. Spreads of human lymphocytes were fixed by ethanol freeze-substitution, digested with ribonuclease and treated at different temperatures prior to staining. Treatment in 0.15 M NaCl-0.0l5 M Na citrate (pH 7.0) for 10 min was followed by 0.15 M NaCl-0.0l5 M Na citrate (pH 7.0) with 10% formalin at the same temperature for 20 min. The GCA staining was localized to nuclei and was measured cell by cell with an integrating scanning cytophotometer. Cellular GCA stain content increased progressively with treatment temperatures above 60°C until 90°C, when it was about 1.5 times that of cells treated at 22°C. Formation of single strand deoxyribonucleic acid at different temperatures was monitored by digesting slides with an endonuclease that is specific for single strand nucleic acids and then staining with GCA. Digestion had little effect on slides that had been treated at less than 70°C, but above this temperature it caused a progressive decrease in stain content up to 90°C, when the stain content was reduced to about one-half that of undigested cells. Both with and without enzymatic digestion, these new approaches to monitoring chromatin denaturation yield preparations that are stable and can be measured on any visible light absorption cytophotometer; in addition, the enzymatic approach exemplifies a class of potential methods in which biochemical procedures are adapted to cytophotometry.

scholarly journals Single-strand nuclease action on heat-denatured spermiogenic chromatin.

1976 ◽  
Vol 24 (8) ◽  
pp. 901-907 ◽  
Author(s):  
J D Hunter ◽  
A J Bodner ◽  
F T Hatch ◽  
R L Balhorn ◽  
J A Mazrimas ◽  
...  
Keyword(s):  
Thermal Denaturation ◽  
Deoxyribonucleic Acid ◽  
Elevated Temperatures ◽  
Chromatin Condensation ◽  
Heat Denaturation ◽  
S1 Nuclease ◽  
Nuclease Digestion ◽  
Aldehyde Groups ◽  
Phosphate Groups

The aim of this study was to compare the sensitivity of chromatin from representative cellular stages of spermiogenesis to a single-strandeded nuclease after heat denaturation. Thermal denaturation of chromatin was assayed in situ in fixed round, elongating and elongated spermatids and in testicular sperm from mice. Production of single-stranded deoxyribonucleic acid (DNA) at elevated temperatures was monitored by digesting chromatin with endonuclease specific for single-stranded DNA (S1 nuclease), staining the residual DNA with gallocyanin-chrome alum (GAC) and measuring the stain content by absorption cytophotometry. Changes in GCA staining were minimal over the temperature range of 22-90 degrees C in each cell type not exposed to nuclease. Staining of undigested cells decreased progressively with advancing cell maturity. Nuclease had no effect on the GCA content of round spermatids below 60 degrees C, but above this temperature there was a progressive decrease in GCA-stainable chromatin. Both round and elongating spermatid stages showed a significantly greater sensitivity to nuclease digestion than did more mature stages; sperm showed no effects of nuclease action below 80 degrees C. Progressive chromatin condensation and a concomitant decrease in the number of available DNA phosphate groups during spermiogenic cell maturation may be responsible for the observed decline in sensitivity to nuclease and decreased GCA staining. Thermal denaturation of round spermatids labeled with 3H-thymidine produced no change in autoradiographic mean nuclear grain counts, indicating no loss of thymidine-labeled DNA from the slides during denaturation. When round spermatids and sperm were hydrolyzed with hot tricholoroacetic acid before staining, both nuclear GCA content and autoradiograph grain count were partially reduced, indicating incomplete DNA removal. Almost complete loss of Feulgen-stainable material occurred in these cells and may be due to depurination and elimination of Feulgren-reactant aldehyde groups.

scholarly journals Cytofluorometric studies on conformation of nucleic acids in situ. II. Denaturation of deoxyribonucleic acid.

1976 ◽  
Vol 24 (1) ◽  
pp. 49-58 ◽  
Author(s):  
Z Darzynkiewicz ◽  
F Traganos ◽  
Z A Arlin ◽  
A T Sharpless ◽  
M R Melamed
Keyword(s):  
Deoxyribonucleic Acid ◽  
Nuclear Dna ◽  
Divalent Cations ◽  
Human Lymphocytes ◽  
Basic Research ◽  
Cell Types ◽  
Leukemic Cells ◽  
Human Leukemia ◽  
Dna Denaturation

Thermal denaturation of deoxyribonucleic acid (DNA) in situ in individual unbroken cells is studied by a cytofluorometric method. This method allows us to investigate DNA denaturation in the presence of divalent cations at concentrations reported to be necessary to maintain native structure of nuclear chromatin. Under these conditions the pattern of DNA denaturation is very different than when studied in the presence of ethylenediaminetetraacetate or citrate. The results suggest that with divalent cations present, the histone basic charges are more uniformly distributed along whole nuclear DNA. Various cell types exhibit great differences in sensitivity to DNA denaturation when assayed in the presence of 1 mM MgCl2. Human lymphocytes, monocytes and certain kinds of human leukemic cells show differences large enough to be used as a parameter for their recognition in mixed samples. Possible applications of the method in basic research on chromatin conformation and as a tool for cell recognition in diagnostic cytology or in the classification of human leukemia are proposed.

scholarly journals BMAP-28, an Antibiotic Peptide of Innate Immunity, Induces Cell Death through Opening of the Mitochondrial Permeability Transition Pore

2002 ◽  
Vol 22 (6) ◽  
pp. 1926-1935 ◽  
Author(s):  
Angela Risso ◽  
Enrico Braidot ◽  
Maria Concetta Sordano ◽  
Angelo Vianello ◽  
Francesco Macrì ◽  
...  
Keyword(s):  
Mitochondrial Permeability Transition Pore ◽  
Mitochondrial Permeability Transition ◽  
Single Cells ◽  
Human Lymphocytes ◽  
Human Tumor ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Inner Mitochondrial Membrane ◽  
Mitochondrial Permeability

ABSTRACT BMAP-28, a bovine antimicrobial peptide of the cathelicidin family, induces membrane permeabilization and death in human tumor cell lines and in activated, but not resting, human lymphocytes. In addition, we found that BMAP-28 causes depolarization of the inner mitochondrial membrane in single cells and in isolated mitochondria. The effect of the peptide was synergistic with that of Ca2+ and inhibited by cyclosporine, suggesting that depolarization depends on opening of the mitochondrial permeability transition pore. The occurrence of a permeability transition was investigated on the basis of mitochondrial permeabilization to calcein and cytochrome c release. We show that BMAP-28 permeabilizes mitochondria to entrapped calcein in a cyclosporine-sensitive manner and that it releases cytochrome c in situ. Our results demonstrate that BMAP-28 is an inducer of the mitochondrial permeability transition pore and that its cytotoxic potential depends on its effects on mitochondrial permeability.

Optics-Free Visualization of Proteins in Single Cells by Time of Flight-Secondary Ion Mass Spectrometry Coupled with Genetically Encoded Chemical Tags

2020 ◽  
Author(s):  
Feifei Jia ◽  
Jie Wang ◽  
Yanyan Zhang ◽  
Qun Luo ◽  
Luyu Qi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Secondary Ion Mass Spectrometry ◽  
Single Cells ◽  
Time Of Flight ◽  
E Coli ◽  
Tof Sims ◽  
Ion Mass Spectrometry ◽  
Chemical Tags ◽  
Secondary Ion

<p></p><p><i>In situ</i> visualization of proteins of interest at single cell level is attractive in cell biology, molecular biology and biomedicine, which usually involves photon, electron or X-ray based imaging methods. Herein, we report an optics-free strategy that images a specific protein in single cells by time of flight-secondary ion mass spectrometry (ToF-SIMS) following genetic incorporation of fluorine-containing unnatural amino acids as a chemical tag into the protein via genetic code expansion technique. The method was developed and validated by imaging GFP in E. coli and human HeLa cancer cells, and then utilized to visualize the distribution of chemotaxis protein CheA in E. coli cells and the interaction between high mobility group box 1 protein and cisplatin damaged DNA in HeLa cells. The present work highlights the power of ToF-SIMS imaging combined with genetically encoded chemical tags for <i>in situ </i>visualization of proteins of interest as well as the interactions between proteins and drugs or drug damaged DNA in single cells.</p><p></p>

In-Situ Dual Beam (FIBSEM) Techniques for Probe Pad Deposition and Dielectric Integrity Inspection in 0.2 μm Technology DRAM Single Cells

1999 ◽  
Author(s):  
Gunnar Zimmermann ◽  
Richard Chapman
Keyword(s):  
Electron Beam ◽  
Single Cell ◽  
Single Cells ◽  
Ion Beam ◽  
Gate Oxide ◽  
Ion Milling ◽  
Dual Beam ◽  
Sem Imaging ◽  
Innovative Techniques

Abstract Dual beam FIBSEM systems invite the use of innovative techniques to localize IC fails both electrically and physically. For electrical localization, we present a quick and reliable in-situ FIBSEM technique to deposit probe pads with very low parasitic leakage (Ipara &lt; 4E-11A at 3V). The probe pads were Pt, deposited with ion beam assistance, on top of highly insulating SiOx, deposited with electron beam assistance. The buried plate (n-Band), p-well, wordline and bitline of a failing and a good 0.2 μm technology DRAM single cell were contacted. Both cells shared the same wordline for direct comparison of cell characteristics. Through this technique we electrically isolated the fail to a single cell by detecting leakage between the polysilicon wordline gate and the cell diffusion. For physical localization, we present a completely in-situ FIBSEM technique that combines ion milling, XeF2 staining and SEM imaging. With this technique, the electrically isolated fail was found to be a hole in the gate oxide at the bad cell.

scholarly journals Effect of temperature, CO2 and O2 on motility and mobility of Anisakidae larvae

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Aiyan Guan ◽  
Inge Van Damme ◽  
Frank Devlieghere ◽  
Sarah Gabriël
Keyword(s):  
Enzymatic Digestion ◽  
Infected Fish ◽  
Effect Of Temperature ◽  
Video Recordings ◽  
Different Temperatures ◽  
Semi Solid ◽  
Zoonotic Parasites ◽  
The Impact ◽  
Phosphate Buffered Saline

AbstractAnisakidae, marine nematodes, are underrecognized fish-borne zoonotic parasites. Studies on factors that could trigger parasites to actively migrate out of the fish are very limited. The objective of this study was to assess the impact of different environmental conditions (temperature, CO2 and O2) on larval motility (in situ movement) and mobility (migration) in vitro. Larvae were collected by candling or enzymatic digestion from infected fish, identified morphologically and confirmed molecularly. Individual larvae were transferred to a semi-solid Phosphate Buffered Saline agar, and subjected to different temperatures (6 ℃, 12 ℃, 22 ℃, 37 ℃) at air conditions. Moreover, different combinations of CO2 and O2 with N2 as filler were tested, at both 6 °C and 12 °C. Video recordings of larvae were translated into scores for larval motility and mobility. Results showed that temperature had significant influence on larval movements, with the highest motility and mobility observed at 22 ℃ for Anisakis spp. larvae and 37 ℃ for Pseudoterranova spp. larvae. During the first 10 min, the median migration of Anisakis spp. larvae was 10 cm at 22 ℃, and the median migration of Pseudoterranova spp. larvae was 3 cm at 37 ℃. Larval mobility was not significantly different under the different CO2 or O2 conditions at 6 °C and 12 ℃. It was concluded that temperature significantly facilitated larval movement with the optimum temperature being different for Anisakis spp. and Pseudoterranova spp., while CO2 and O2 did not on the short term. This should be further validated in parasite-infected/spiked fish fillets.

scholarly journals A Superhydrophobic, Antibacterial, and Durable Surface of Poplar Wood

Nanomaterials ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1885
Author(s):  
Xinyu Wu ◽  
Feng Yang ◽  
Jian Gan ◽  
Zhangqian Kong ◽  
Yan Wu
Keyword(s):  
Stearic Acid ◽  
Antibacterial Properties ◽  
Alkali Resistance ◽  
Poplar Wood ◽  
X Ray Diffraction ◽  
X Ray ◽  
Different Temperatures ◽  
Acid And Alkali Resistance ◽  
The Stability

The silver particles were grown in situ on the surface of wood by the silver mirror method and modified with stearic acid to acquire a surface with superhydrophobic and antibacterial properties. Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and X-ray energy spectroscopy (XPS) were used to analyze the reaction mechanism of the modification process. Scanning electron microscopy (SEM) and contact angle tests were used to characterize the wettability and surface morphology. A coating with a micro rough structure was successfully constructed by the modification of stearic acid, which imparted superhydrophobicity and antibacterial activity to poplar wood. The stability tests were performed to discuss the stability of its hydrophobic performance. The results showed that it has good mechanical properties, acid and alkali resistance, and UV stability. The durability tests demonstrated that the coating has the function of water resistance and fouling resistance and can maintain the stability of its hydrophobic properties under different temperatures of heat treatment.

scholarly journals Label-Free and Quantitative Dry Mass Monitoring for Single Cells during In Situ Culture

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1635
Author(s):  
Ya Su ◽  
Rongxin Fu ◽  
Wenli Du ◽  
Han Yang ◽  
Li Ma ◽  
...  
Keyword(s):  
Cell Growth ◽  
Single Cell ◽  
Hela Cells ◽  
Quantitative Measurement ◽  
Single Cells ◽  
Dry Mass ◽  
Quantitative Detection ◽  
Label Free ◽  
Mass Sensitivity

Quantitative measurement of single cells can provide in-depth information about cell morphology and metabolism. However, current live-cell imaging techniques have a lack of quantitative detection ability. Herein, we proposed a label-free and quantitative multichannel wide-field interferometric imaging (MWII) technique with femtogram dry mass sensitivity to monitor single-cell metabolism long-term in situ culture. We demonstrated that MWII could reveal the intrinsic status of cells despite fluctuating culture conditions with 3.48 nm optical path difference sensitivity, 0.97 fg dry mass sensitivity and 2.4% average maximum relative change (maximum change/average) in dry mass. Utilizing the MWII system, different intrinsic cell growth characteristics of dry mass between HeLa cells and Human Cervical Epithelial Cells (HCerEpiC) were studied. The dry mass of HeLa cells consistently increased before the M phase, whereas that of HCerEpiC increased and then decreased. The maximum growth rate of HeLa cells was 11.7% higher than that of HCerEpiC. Furthermore, HeLa cells were treated with Gemcitabine to reveal the relationship between single-cell heterogeneity and chemotherapeutic efficacy. The results show that cells with higher nuclear dry mass and nuclear density standard deviations were more likely to survive the chemotherapy. In conclusion, MWII was presented as a technique for single-cell dry mass quantitative measurement, which had significant potential applications for cell growth dynamics research, cell subtype analysis, cell health characterization, medication guidance and adjuvant drug development.

scholarly journals Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2206
Author(s):  
Thai Pham ◽  
Renjie Liao ◽  
Joshua Labaer ◽  
Jia Guo
Keyword(s):  
High Performance ◽  
Single Cells ◽  
Protein Quantification ◽  
Cellular Systems ◽  
Protein Profiling ◽  
High Signal ◽  
Protein Targets ◽  
Chemical Reagents ◽  
Ffpe Tissues

Understanding the composition, function and regulation of complex cellular systems requires tools that quantify the expression of multiple proteins at their native cellular context. Here, we report a highly sensitive and accurate protein in situ profiling approach using off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this method, protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and CFT. Subsequently, the fluorophores are efficiently cleaved by mild chemical reagents, which simultaneously deactivate HRP. Through reiterative cycles of protein staining, fluorescence imaging, fluorophore cleavage, and HRP deactivation, multiplexed protein quantification in single cells in situ can be achieved. We designed and synthesized the high-performance CFT, and demonstrated that over 95% of the staining signals can be erased by mild chemical reagents while preserving the integrity of the epitopes on protein targets. Applying this method, we explored the protein expression heterogeneity and correlation in a group of genetically identical cells. With the high signal removal efficiency, this approach also enables us to accurately profile proteins in formalin-fixed paraffin-embedded (FFPE) tissues in the order of low to high and also high to low expression levels.

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