scholarly journals A simple method for freeze-fracture of monolayer cultures.

1975 ◽  
Vol 67 (3) ◽  
pp. 904-911 ◽  
Author(s):  
T R Collins ◽  
J C Bartholomew ◽  
M Calvin
Keyword(s):  
Freeze Fracture ◽  
Silicon Monoxide ◽  
Cellular Interaction ◽  
3T3 Cells ◽  
Simple Method ◽  
Mouse Embryo Fibroblasts ◽  
Membrane Specialization ◽  
Monolayer Cultures ◽  
Growth Properties

A simple method is described for the freeze-fracture in situ of monolayer cultures grown on gold carriers coated with a thin layer of silicon monoxide. Preliminary observations on 3T3 mouse embryo fibroblasts indicate that this technique exposes large areas of cell membrane, making it possible to determine how areas of membrane specialization are related to the cell as a whole and to regions of cellular interaction. 3T3 cells cultured on silicon monoxide show no modification of growth properties compared to cells growing on Falcon plastic, and other cell lines also appear to grow well on this substrate.

scholarly journals Specializations in filopodial membranes at points of attachment to the substrate.

1980 ◽  
Vol 87 (3) ◽  
pp. 562-568 ◽  
Author(s):  
J M Robinson ◽  
M J Karnovsky
Keyword(s):  
Cell Body ◽  
Freeze Fracture ◽  
Spatial Relationship ◽  
Cholesterol Content ◽  
Mouse Cell ◽  
Striking Difference ◽  
Functional Specialization ◽  
Monolayer Cultures ◽  
Relationship Of

A mouse cell line (LM), which grows predominantly as spindle-shaped cells with numerous filopodia, was employed in this study. These filopodial projections appear to be important as sites of attachment to the substratum in LM cells. Morphologically the filopodia are slender projections from the cell body which usually attach to the substrate at their distal ends (filopodial footpads). Freeze-fracture of monolayer cultures in situ preserves the spatial relationship of filopodial processes to that of the cell body. Examination of these freeze-fracture preparations reveals a striking difference in the density of intramembrane particles (IMP) in the filopodial-footpad plasmalemma compared with the plasmalemma of the cell body (number of IMP in footpad > cell body). Additionally, there is a marked difference in the number of filipin-sterol complexes on the cell body, compared with the filopodial footpad, implying a difference in the cholesterol content in these regions (filipin-sterol complexes in footpad < cell body). These data suggest a structural and functional specialization of the filopodial-footpad plasma membrane which may be related to cell adhesion.

scholarly journals Freeze-fracture of monolayer cultures.

1977 ◽  
Vol 72 (3) ◽  
pp. 763-769 ◽  
Author(s):  
B Pauli ◽  
R S Weinstein ◽  
L W Soble ◽  
J Alroy
Keyword(s):  
Tissue Culture ◽  
Organ Culture ◽  
Culture System ◽  
Freeze Fracture ◽  
Tissue Cultures ◽  
High Quality ◽  
Simple Method ◽  
Freeze Fracturing ◽  
Organ Culture System ◽  
Monolayer Cultures

This paper describes a simple method for the freeze-fracturing of cells in monolayers or multi-layer tissue cultures. The method produces high quality replicas and is applicable to the study of virtually any tissue culture or organ culture system. It uses standard materials and equipment for both tissue culture and freeze-fracturing.

Rotary Beveling for In Situ Thin-Section Microscopy of Cell Cultures

1981 ◽  
Vol 39 ◽  
pp. 550-551
Author(s):  
Dean A. Handley ◽  
Jack T. Alexander ◽  
Shu Chien
Keyword(s):  
Cell Growth ◽  
Cell Cultures ◽  
Molecular Interactions ◽  
Convenient Method ◽  
Petri Dish ◽  
Simple Method ◽  
Thin Section Microscopy ◽  
Thin Sectioning ◽  
Organic Fluids

In situ preparation of cell cultures for ultrastructural investigations is a convenient method by which fixation, dehydration and embedment are carried out in the culture petri dish. The in situ method offers the advantage of preserving the native orientation of cell-cell interactions, junctional regions and overlapping configurations. In order to section after embedment, the petri dish is usually separated from the polymerized resin by either differential cryo-contraction or solvation in organic fluids. The remaining resin block must be re-embedded before sectioning. Although removal of the petri dish may not disrupt the native cellular geometry, it does sacrifice what is now recognized as an important characteristic of cell growth: cell-substratum molecular interactions. To preserve the topographic cell-substratum relationship, we developed a simple method of tapered rotary beveling to reduce the petri dish thickness to a dimension suitable for direct thin sectioning.

A Mild and Simple Method for Making MIDA Boronates

2020 ◽  
Author(s):  
Aidan Kelly ◽  
Peng-Jui (Ruby) Chen ◽  
Jenna Klubnick ◽  
Daniel J. Blair ◽  
Martin D. Burke
Keyword(s):  
Organic Synthesis ◽  
Boronic Acids ◽  
Simple Method ◽  
Direct Preparation ◽  
Synthesis Procedure ◽  
Harsh Conditions

<div> <div> <div> <p>Existing methods for making MIDA boronates require harsh conditions and complex procedures to achieve dehydration. Here we disclose that a pre-dried form of MIDA, MIDA anhydride, acts as both a source of the MIDA ligand and an in situ desiccant to enable a mild and simple MIDA boronate synthesis procedure. This method expands the range of sensitive boronic acids that can be converted into their MIDA boronate counterparts. Further utilizing unique properties of MIDA boronates, we have developed a MIDA Boronate Maker Kit which enables the direct preparation and purification of MIDA boronates from boronic acids using only heating and centrifuge equipment that is widely available in labs that do not specialize in organic synthesis. </p> </div> </div> </div>

scholarly journals Assessing an Atmospheric Correction Algorithm for Time Series of Satellite-Based Water-Leaving Reflectance Using Match-Up Sites in Australian Coastal Waters

2021 ◽  
Vol 13 (10) ◽  
pp. 1927
Author(s):  
Fuqin Li ◽  
David Jupp ◽  
Thomas Schroeder ◽  
Stephen Sagar ◽  
Joshua Sixsmith ◽  
...  
Keyword(s):  
Coastal Waters ◽  
Atmospheric Correction ◽  
Correction Method ◽  
In Situ Measurements ◽  
Landsat 8 ◽  
Correction Algorithm ◽  
Simple Method ◽  
North East ◽  
The Impact

An atmospheric correction algorithm for medium-resolution satellite data over general water surfaces (open/coastal, estuarine and inland waters) has been assessed in Australian coastal waters. In situ measurements at four match-up sites were used with 21 Landsat 8 images acquired between 2014 and 2017. Three aerosol sources (AERONET, MODIS ocean aerosol and climatology) were used to test the impact of the selection of aerosol optical depth (AOD) and Ångström coefficient on the retrieved accuracy. The initial results showed that the satellite-derived water-leaving reflectance can have good agreement with the in situ measurements, provided that the sun glint is handled effectively. Although the AERONET aerosol data performed best, the contemporary satellite-derived aerosol information from MODIS or an aerosol climatology could also be as effective, and should be assessed with further in situ measurements. Two sun glint correction strategies were assessed for their ability to remove the glint bias. The most successful one used the average of two shortwave infrared (SWIR) bands to represent sun glint and subtracted it from each band. Using this sun glint correction method, the mean all-band error of the retrieved water-leaving reflectance at the Lucinda Jetty Coastal Observatory (LJCO) in north east Australia was close to 4% and unbiased over 14 acquisitions. A persistent bias in the other strategy was likely due to the sky radiance being non-uniform for the selected images. In regard to future options for an operational sun glint correction, the simple method may be sufficient for clear skies until a physically based method has been established.

scholarly journals 762 A combination of functional biomarkers improves identification of the tumor-specific reactive T cell repertoire

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A810-A810
Author(s):  
Arianna Draghi ◽  
Katja Harbst ◽  
Inge Svane ◽  
Marco Donia
Keyword(s):  
T Cells ◽  
T Cell ◽  
De Novo ◽  
Autologous Tumor ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Simple Method ◽  
Functional Biomarkers

BackgroundDetecting the entire repertoire of tumor-specific reactive T cells is essential for investigating the broad range of T cell functions in the tumor-microenvironment. At present, assays identifying tumor-specific functional activation measure either upregulation of specific surface molecules, de novo production of the most common antitumor cytokines or mobilization of cytotoxic granules.MethodsIn this study, we combined transcriptomic analyses of tumor-specific reactive tumorinfiltrating lymphocytes (TILs), TIL-autologous tumor cell co-cultures and commonly used established detection protocols to develop an intracellular flow cytometry staining method encompassing simultaneous detection of intracellular CD137, de novo production of TNF and IFNy and extracellular mobilization of CD107a.ResultsThis approach enabled the identification of a larger fraction of tumor-specific reactive T cells in vitro compared to standard methods, revealing the existence of multiple distinct functional clusters of tumor-specific reactive TILs. Publicly available datasets of fresh tumor single-cell RNA-sequencing from four cancer types were investigated to confirm that these functional biomarkers identified distinct functional clusters forming the entire repertoire of tumor-specific reactive T cells in situ.ConclusionsIn conclusion, we describe a simple method using a combination of functional biomarkers that improves identification of the tumor-specific reactive T cell repertoire in vitro and in situ.

scholarly journals Graphene Oxide-Embedded Extracellular Matrix- Derived Hydrogel as a Multiresponsive Platform for 3D Bioprinting Applications

2021 ◽  
Vol 7 (3) ◽  
Author(s):  
Laura Rueda-Gensini ◽  
Julian A Serna ◽  
Javier Cifuentes ◽  
Juan C Cruz ◽  
Carolina Muñoz-Camargo
Keyword(s):  
Graphene Oxide ◽  
Mechanical Stability ◽  
Light Exposure ◽  
Structural Characteristics ◽  
Elastic Response ◽  
Cellular Interaction ◽  
3D Bioprinting ◽  
In Situ Reduction ◽  
Crosslinking Degree

Decellularized extracellular matrices (dECMs) have shown enormous potential for the biofabrication of tissues due to their biomimetic properties that promote enhanced cellular interaction and tissue regeneration. However, biofabrication schemes requiring electrostimulation pose an additional constraint due to the insulating properties of natural materials. Here, we propose a methacryloyl-modified decellularized small intestine submucosa (SISMA) hydrogel, embedded with graphene oxide (GO) nanosheets, for extrusion-based 3D bioprinting applications that require electrostimulation. Methacryloyl biochemicalmodification is performed to enhance the mechanical stability of dECM constructs by mediating photo-crosslinking reactions, and a multistep fabrication scheme is proposed to harness the bioactive and hydrophilic properties of GO and electroconductive properties of reduced GO. For this, GO was initially dispersed in SISMA hydrogels by exploiting its hydrophilicity and protein adsorption capabilities, and in situ reduction was subsequently performed to confer electroconductive abilities. SISMA-GO composite hydrogels were successfully prepared with enhanced structural characteristics, as shown by the higher crosslinking degree and increased elastic response upon blue-light exposure. Moreover, GO was homogeneously dispersed without affecting photocrosslinking reactions and hydrogel shear-thinning properties. Human adipose-derived mesenchymal stem cells were successfully bioprinted in SISMA-GO with high cell viability after 1 week and in situ reduction of GO during this period enhanced the electrical conductivity of these nanostructures. This work demonstrates the potential of SISMA-GO bioinks as bioactive and electroconductive scaffolds for electrostimulation applications in tissue engineering and regenerative medicine.

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