scholarly journals Glass beads load macromolecules into living cells

1987 ◽  
Vol 88 (5) ◽  
pp. 669-678
Author(s):  
P.L. McNeil ◽  
E. Warder
Keyword(s):  
Culture Medium ◽  
Small Volume ◽  
Cultured Cells ◽  
Cell Monolayer ◽  
Cell Loss ◽  
Living Cells ◽  
Glass Beads ◽  
Cell Type ◽  
Large Numbers ◽  
Micron Diameter

We describe and characterize an exceptionally rapid and simple new technique for loading large numbers of cultured cells with large macromolecules. The culture medium of the cell monolayer is replaced by a small volume of the macromolecule to be loaded. Glass beads (75–500 micron diameter) are then sprinkled onto the cells, the cells are washed free of beads and exogenous macromolecules, and ‘bead-loading’ is completed. The conditions for bead-loading can readily be modified to accommodate cell type and loading objectives: for example, the amount of loading per cell increases if bead size is increased or if beads are agitated after sprinkling onto the monolayer, but at the expense of increased cell loss. As many as 97% of a population of bovine aortic endothelial (BAE) cells were loaded with a 10,000 Mr dextran; and 79% with a 150,000 Mr dextran using bead-loading. Various cell lines have been loaded using glass beads. Moreover, bead-loading has the advantage of producing loaded cells that remain adherent and well-spread, thus minimizing recovery time and permitting immediate microscopic examination.

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

Improved micro-impedance spectroscopy to determine cell barrier properties

2020 ◽  
Vol 4 (3) ◽  
pp. 150-155 ◽  
Author(s):  
Md. Mehadi Hasan Sohag ◽  
Olivier Nicoud ◽  
Racha Amine ◽  
Abir Khalil-Mgharbel ◽  
Jean-Pierre Alcaraz ◽  
...  
Keyword(s):  
Impedance Spectroscopy ◽  
Epithelial Cells ◽  
Lipid Bilayers ◽  
Cultured Cells ◽  
Cell Model ◽  
Measurement Techniques ◽  
Cell Monolayer ◽  
Microfluidic Channel ◽  
Small Surface ◽  
Resistance Pathway

AbstractThe goal of this study was to determine whether the Tethapod system, which was designed to determine the impedance properties of lipid bilayers, could be used for cell culture in order to utilise micro-impedance spectroscopy to examine further biological applications. To that purpose we have used normal epithelial cells from kidney (RPTEC) and a kidney cancer cell model (786-O). We demonstrate that the Tethapod system is compatible with the culture of 10,000 cells seeded to grow on a small area gold measurement electrode for several days without affecting the cell viability. Furthermore, the range of frequencies for EIS measurements were tuned to examine easily the characteristics of the cell monolayer. We demonstrate significant differences in the paracellular resistance pathway between normal and cancer kidney epithelial cells. Thus, we conclude that this device has advantages for the study of cultured cells that include (i) the configuration of measurement and reference electrodes across a microfluidic channel, and (ii) the small surface area of 6 parallel measurement electrodes (2.1 mm2) integrated in a microfluidic system. These characteristics might improve micro-impedance spectroscopy measurement techniques to provide a simple tool for further studies in the field of the patho-physiology of biological barriers.

Nitrification: an old process, a new concern

1971 ◽  
Vol 24 ◽  
Author(s):  
W. H. Verstraete
Keyword(s):  
Culture Medium ◽  
Nitrogen Sources ◽  
Living Cells ◽  
Factors Affecting ◽  
Inhibiting Effect ◽  
Proteose Peptone ◽  
Asparaginase Activity

Some  factors affecting the L-asparaginase activity of E.  aroideae were investigated. Increasing  concentrations of glucose in the culture medium had an inhibiting effect on  the production of L-asparaginase by this microorganism. Buffering of the  culture medium in order to stabilize the pH during growth resulted in a decrease  of the L-asparaginase activity. From the different nitrogen sources examined,  tryptone, proteose peptone nr 2 and nr 3 stimulated the L-asparaginase  production. Toluene treatment of the cells practically destroyed the  L-asparaginase. Acetone dried cells showed an L-asparaginase activity  comparable with the activity of living cells.

scholarly journals A Setup for Microscopic Studies of Ultrasounds Effects on Microliters Scale Samples: Analytical, Numerical and Experimental Characterization

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 847
Author(s):  
Florian N. Gailliègue ◽  
Mindaugas Tamošiūnas ◽  
Franck M. André ◽  
Lluis M. Mir
Keyword(s):  
Culture Medium ◽  
Small Volume ◽  
Acoustic Pressure ◽  
Experimental Characterization ◽  
Focal Distance ◽  
Microscopic Studies ◽  
Cell Membrane Permeabilization ◽  
Ultrasonic Parameters ◽  
Rigid Walls

Sonoporation is the process of cell membrane permeabilization, due to exposure to ultrasounds. There is a lack of consensus concerning the mechanisms of sonoporation: Understanding the mechanisms of sonoporation refines the choice of the ultrasonic parameters to be applied on the cells. Cells’ classical exposure systems to ultrasounds have several drawbacks, like the immersion of the cells in large volumes of liquid, the nonhomogeneous acoustic pressure in the large sample, and thus, the necessity for magnetic stirring to somehow homogenize the exposure of the cells. This article reports the development and characterization of a novel system allowing the exposure to ultrasounds of very small volumes and their observation under the microscope. The observation under a microscope imposes the exposure of cells and Giant Unilamellar Vesicles under an oblique incidence, as well as the very unusual presence of rigid walls limiting the sonicated volume. The advantages of this new setup are not only the use of a very small volume of cells culture medium/microbubbles (MB), but the presence of flat walls near the sonicated region that results in a more homogeneous ultrasonic pressure field, and thus, the control of the focal distance and the real exposure time. The setup presented here comprises the ability to survey the geometrical and dynamical aspects of the exposure of cells and MB to ultrasounds, if an ultrafast camera is used. Indeed, the setup thus fulfills all the requirements to apply ultrasounds conveniently, for accurate mechanistic experiments under an inverted fluorescence microscope, and it could have interesting applications in photoacoustic research.

scholarly journals Assembly of vimentin in cultured cells varies with cell type

1989 ◽  
Vol 264 (30) ◽  
pp. 17953-17960
Author(s):  
W B Isaacs ◽  
R K Cook ◽  
J C Van Atta ◽  
C M Redmond ◽  
A B Fulton
Keyword(s):  
Cultured Cells ◽  
Cell Type

Downregulation of renal TonEBP in hypokalemic rats

2007 ◽  
Vol 293 (1) ◽  
pp. F408-F415 ◽  
Author(s):  
Un Sil Jeon ◽  
Ki-Hwan Han ◽  
Soo-Hyun Park ◽  
Sang Do Lee ◽  
Mee Rie Sheen ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Renal Medulla ◽  
Distal Tubule ◽  
Cell Type ◽  
Outer Medulla ◽  
Sodium Transporters ◽  
Enhancer Binding Protein ◽  
Collecting Ducts ◽  
Cell Type Specific ◽  
Underlying Mechanisms

Hypokalemia causes a significant decrease in the tonicity of the renal medullary interstitium in association with reduced expression of sodium transporters in the distal tubule. We asked whether hypokalemia caused downregulation of the tonicity-responsive enhancer binding protein (TonEBP) transcriptional activator in the renal medulla due to the reduced tonicity. We found that the abundance of TonEBP decreased significantly in the outer and inner medullas of hypokalemic rats. Underlying mechanisms appeared different in the two regions because the abundance of TonEBP mRNA was lower in the outer medulla but unchanged in the inner medulla. Immunohistochemical examination of TonEBP revealed cell type-specific differences. TonEBP expression decreased dramatically in the outer and inner medullary collecting ducts, thick ascending limbs, and interstitial cells. In the descending and ascending thin limbs, TonEBP abundance decreased modestly. In the outer medulla, TonEBP shifted to the cytoplasm in the descending thin limbs. As expected, transcription of aldose reductase, a target of TonEBP, was decreased since the abundance of mRNA and protein was reduced. Downregulation of TonEBP appeared to have also contributed to reduced expression of aquaporin-2 and UT-A urea transporters in the renal medulla. In cultured cells, expression and activity of TonEBP were not affected by reduced potassium concentrations in the medium. These data support the view that medullary tonicity regulates expression and nuclear distribution of TonEBP in the renal medulla in cell type-specific manners.

MIC-Drop: A platform for large-scale in vivo CRISPR screens

Science ◽  
2021 ◽  
pp. eabi8870
Author(s):  
Saba Parvez ◽  
Chelsea Herdman ◽  
Manu Beerens ◽  
Korak Chakraborti ◽  
Zachary P. Harmer ◽  
...  
Keyword(s):  
Large Scale ◽  
Cultured Cells ◽  
Cardiac Development ◽  
Droplet Microfluidics ◽  
Model Organisms ◽  
Genetic Screens ◽  
Large Numbers ◽  
And Function ◽  
Genome Scale

CRISPR-Cas9 can be scaled up for large-scale screens in cultured cells, but CRISPR screens in animals have been challenging because generating, validating, and keeping track of large numbers of mutant animals is prohibitive. Here, we report Multiplexed Intermixed CRISPR Droplets (MIC-Drop), a platform combining droplet microfluidics, single-needle en masse CRISPR ribonucleoprotein injections, and DNA barcoding to enable large-scale functional genetic screens in zebrafish. The platform can efficiently identify genes responsible for morphological or behavioral phenotypes. In one application, we show MIC-Drop can identify small molecule targets. Furthermore, in a MIC-Drop screen of 188 poorly characterized genes, we discover several genes important for cardiac development and function. With the potential to scale to thousands of genes, MIC-Drop enables genome-scale reverse-genetic screens in model organisms.

scholarly journals CYTOPLASMIC DNA SYNTHESIS IN AMOEBA PROTEUS

1962 ◽  
Vol 15 (3) ◽  
pp. 525-534 ◽  
Author(s):  
M. Rabinovitch ◽  
W. Plaut
Keyword(s):  
Dna Synthesis ◽  
Tritiated Thymidine ◽  
Amoeba Proteus ◽  
Living Cells ◽  
Large Numbers ◽  
Cytoplasmic Dna ◽  
Basic Dyes ◽  
Nuclease Digestion ◽  
High Degree ◽  
Very High

The incorporation of tritiated thymidine in Amoeba proteus was reinvestigated in order to see if it could be associated with microscopically detectable structures. Staining experiments with basic dyes, including the fluorochrome acridine orange, revealed the presence of large numbers of 0.3 to 0.5 µ particles in the cytoplasm of all cells studied. The effect of nuclease digestion on the dye affinity of the particles suggests that they contain DNA as well as RNA. Centrifugation of living cells at 10,000 g leads to the sedimentation of the particles in the centrifugal third of the ameba near the nucleus. Analysis of centrifuged cells which had been incubated with H3-thymidine showed a very high degree of correlation between the location of the nucleic acid-containing granules and that of acid-insoluble, deoxyribonuclease-sensitive labeled molecules and leads to the conclusion that cytoplasmic DNA synthesis in Amoeba proteus occurs in association with these particles.

scholarly journals Super-cells untangle large and complex single-cell transcriptome networks

2021 ◽  
Author(s):  
Mariia Bilous ◽  
Loc Tran ◽  
Chiara Cianciaruso ◽  
Santiago J Carmona ◽  
Mikael J Pittet ◽  
...  
Keyword(s):  
Single Cell ◽  
Coarse Graining ◽  
Cell Populations ◽  
Cell Type ◽  
Large Numbers ◽  
Single Cell Rna Sequencing ◽  
Gene Correlation ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

Single-cell RNA sequencing (scRNA-seq) technologies offer unique opportunities for exploring heterogeneous cell populations. However, in-depth single-cell transcriptomic characterization of complex tissues often requires profiling tens to hundreds of thousands of cells. Such large numbers of cells represent an important hurdle for downstream analyses, interpretation and visualization. Here we develop a network-based coarse-graining framework where highly similar cells are merged into super-cells. We demonstrate that super-cells not only preserve but often improve the results of downstream analyses including visualization, clustering, differential expression, cell type annotation, gene correlation, imputation, RNA velocity and data integration. By capitalizing on the redundancy inherent to scRNA-seq data, super-cells significantly facilitate and accelerate the construction and interpretation of single-cell atlases, as demonstrated by the integration of 1.46 million cells from COVID-19 patients in less than two hours on a standard desktop.

The Immunohistochemical Characterisation of an SV40-immortalised Human Corneal Epithelial Cell Line

2003 ◽  
Vol 31 (4) ◽  
pp. 409-417 ◽  
Author(s):  
Anne Huhtala ◽  
Sami K. Nurmi ◽  
Hanna Tähti ◽  
Lotta Salminen ◽  
Päivi Alajuuma ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Single Cell ◽  
Cell Line ◽  
Cell Cultures ◽  
Corneal Epithelium ◽  
Culture Medium ◽  
Corneal Epithelial Cell ◽  
Cell Type ◽  
Corneal Epithelial ◽  
Single Cell Type

Alternatives to the Draize rabbit eye irritation test are currently being investigated. Because of morphological and biochemical differences between the rabbit and the human eye, continuous human cell lines have been proposed for use in ocular toxicology studies. Single cell-type monolayer cultures in culture medium have been used extensively in ocular toxicology. In the present study, an SV40-immortalised human corneal epithelial (HCE) cell line was characterised immunohistochemically, by using 13 different monoclonal antibodies to cytokeratins (CKs), ranging from CK3 to CK20. The results from the monolayer HCE cell cultures were compared with those from the corneal epithelium of human corneal cryostat sections. Previous studies have shown that the morphology of the HCE cell is similar to that of primary cultured human corneal epithelial cells, and that the cells express the cornea-specific CK3. In the study reported here, we show that the cell line also expresses CKs 7, 8, 18 and 19. These CKs are typically expressed by simple epithelial cells, and are not found in the human cornea in vivo. Therefore, the monolayer HCE cell line grown in culture medium does not express the CK pattern that is typical of human corneal epithelium. This should be taken into consideration when using HCE cell cultures in similar single cell-type experiments for ocular toxicology.

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