scholarly journals Interfacing Live Cells with Surfaces: A Concurrent Control Technique for Quantifying Surface Ligand Activity

2021 ◽  
Author(s):  
Michael C. Robitaille ◽  
Joseph A. Christodoulides ◽  
Patrick Calhoun ◽  
Jeff M. Byers ◽  
Marc P. Raphael
Keyword(s):  
Cellular Adhesion ◽  
Live Cell ◽  
Control Technique ◽  
Live Cells ◽  
Chip Surface ◽  
Concurrent Control ◽  
Activity Measurements ◽  
Surface Ligand ◽  
Ligand Activity

AbstractSurface ligand activity is a key design parameter for successfully interfacing surfaces with cells - whether in the context of in vitro investigations for understanding cellular signaling pathways or more applied applications in drug delivery and medical implants. Unlike other crucial surface parameters, such as stiffness and roughness, surface ligand activity currently lacks a standardized measurement approach that can be readily paired with live cell investigations. To fill this void, we have developed a concurrent control technique for characterizing in vitro ligand surface activity. Pairs of gold-coated glass chips were biofunctionalized with RGD ligand in a parallel workflow: one chip for in vitro applications and the other for surface plasmon resonance (SPR) based RGD activity characterization. Recombinant αVβ3 integrins were injected over the SPR chip surface as mimics of the cellular membrane bound receptors and the resulting binding kinetics parameterized to quantify ligand activity. These activity measurements were correlated with cell morphological features, measured by interfacing MDA-MB-231 cells with the in vitro chip surfaces on the live cell microscope. We show that the SPR concurrent control approach has multiple advantages based on the facts that SPR is a standardized technique and has the sensitivity to measure ligand activity across the most relevant range of extracellular surface densities. Furthermore, by pairing both SPR and in vitro approaches, a comparison of the results can provide biological insights into the nature of cellular adhesion and dynamics.

scholarly journals Interfacing Live Cells with Surfaces: A Concurrent Control Technique for Quantifying Surface Ligand Activity

2021 ◽  
Author(s):  
Michael C. Robitaille ◽  
Joseph A. Christodoulides ◽  
Patrick J. Calhoun ◽  
Jeff M. Byers ◽  
Marc P. Raphael
Keyword(s):  
Control Technique ◽  
Live Cells ◽  
Concurrent Control ◽  
Surface Ligand ◽  
Ligand Activity

scholarly journals tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

2021 ◽  
Author(s):  
Y. Bousmah ◽  
H. Valenta ◽  
G. Bertolin ◽  
U. Singh ◽  
V. Nicolas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy ◽  
Live Cell ◽  
Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

Effects of medium and hypothermic temperatures on preservation of isolated porcine testis cells

2010 ◽  
Vol 22 (3) ◽  
pp. 523 ◽  
Author(s):  
Yanfei Yang ◽  
Ali Honaramooz
Keyword(s):  
Trypan Blue ◽  
Cell Isolation ◽  
Live Cell ◽  
Live Cells ◽  
Cell Recovery ◽  
Trypan Blue Exclusion ◽  
Culture Studies ◽  
Porcine Testis ◽  
Peritubular Cells

The effects of medium and hypothermic temperatures on testis cells were investigated to develop a strategy for their short-term preservation. Testes from 1-week-old piglets were enzymatically dissociated for cell isolation. In Experiment 1, testis cells were stored at either room (RT) or refrigeration (RG) temperature for 6 days in one of 13 different media. Live cell recovery was assayed daily using trypan blue exclusion. In Experiment 2, three media at RG were selected for immunocytochemical and in vitro culture studies. Live cell recovery was also assayed daily for 6 days using both trypan blue exclusion and a fluorochrome assay kit. For all media tested, significantly or numerically more live cells were maintained at RG than RT. On preservation Day 3 at RG (cell isolation day as Day 0), 20% FBS-Leibovitz resulted in the highest live cell recovery (89.5 ± 1.7%) and DPBS in the lowest (60.3 ± 1.9%). On Day 6 at RG, 20% FBS- Leibovitz also resulted in the best preservation efficiency with 80.9 ± 1.8% of Day 0 live cells recovered. There was no difference in live cell recovery detected by the two viability assays. After preservation, the proportion of gonocytes did not change, whereas that of Sertoli and peritubular cells increased and decreased, respectively. After 6 days of hypothermic preservation, testis cells showed similar culture potential to fresh cells. These results show that testis cells can be preserved for 6 days under hypothermic conditions with a live cell recovery of more than 80% and after-storage viability of 88%.

Ligand-displacement-based two-photon fluorogenic probe for visualizing mercapto biomolecules in live cells, Drosophila brains and zebrafish

The Analyst ◽  
2018 ◽  
Vol 143 (14) ◽  
pp. 3433-3441 ◽  
Author(s):  
Yanfei Zhao ◽  
Yun Ni ◽  
Liulin Wang ◽  
Chenchen Xu ◽  
Chenqi Xin ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Live Cell ◽  
Live Cells ◽  
Two Photon ◽  
Ligand Displacement ◽  
Cell Tissue ◽  
Fluorogenic Probe ◽  
Displacement Based

We report the Fe(iii)-based complex TPFeS which acts as a novel ligand-displacement-based TP fluorogenic probe for the rapid detection of mercapto biomolecules both in vitro and in live cell/tissue/in vivo imaging.

scholarly journals Specific GFP-binding artificial proteins (αRep): a new tool for in vitro to live cell applications

2015 ◽  
Vol 35 (4) ◽  
Author(s):  
Anne Chevrel ◽  
Agathe Urvoas ◽  
Ines Li de la Sierra-Gallay ◽  
Magali Aumont-Nicaise ◽  
Sandrine Moutel ◽  
...  
Keyword(s):  
Live Cell ◽  
Live Cells ◽  
Artificial Proteins

Artificial proteins, named αRep, binding tightly and specifically to EGFP are described. The structures of αRep–EGFP complexes explain how αRep recognize their cognate partner. Specific αRep can be used for biochemical or live cells experiments.

Recognition of saccharides in the NIR region with a novel fluorogenic boronolectin: in vitro and live cell labeling

2015 ◽  
Vol 51 (23) ◽  
pp. 4895-4898 ◽  
Author(s):  
Cecilia Samaniego Lopez ◽  
María Amparo Lago Huvelle ◽  
María Laura Uhrig ◽  
Federico Coluccio Leskow ◽  
Carla C. Spagnuolo
Keyword(s):  
Acid Derivative ◽  
Boronic Acid ◽  
Near Infrared ◽  
Detection Performance ◽  
Live Cell ◽  
Cell Labeling ◽  
Live Cells ◽  
Live Cell Labeling

The detection performance in solution and in live cells of a novel mono-boronic acid derivative of a near-infrared luminescent tricarbocyanine with OFF–ON response upon addition of saccharides.

scholarly journals Current Development in Interdigital Transducer (IDT) Surface Acoustic Wave Devices for Live Cell In Vitro Studies: A Review

Micromachines ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 30
Author(s):  
Mazlee Bin Mazalan ◽  
Anas Mohd Noor ◽  
Yufridin Wahab ◽  
Shuhaida Yahud ◽  
Wan Safwani Wan Kamarul Zaman
Keyword(s):  
Acoustic Waves ◽  
Surface Acoustic Waves ◽  
Microfluidic System ◽  
Live Cell ◽  
Cell Manipulation ◽  
Live Cells ◽  
Future Application ◽  
Wide Range ◽  
Separation Cell

Acoustics have a wide range of uses, from noise-cancelling to ultrasonic imaging. There has been a surge in interest in developing acoustic-based approaches for biological and biomedical applications in the last decade. This review focused on the application of surface acoustic waves (SAW) based on interdigital transducers (IDT) for live-cell investigations, such as cell manipulation, cell separation, cell seeding, cell migration, cell characteristics, and cell behaviours. The approach is also known as acoustofluidic, because the SAW device is coupled with a microfluidic system that contains live cells. This article provides an overview of several forms of IDT of SAW devices on recently used cells. Conclusively, a brief viewpoint and overview of the future application of SAW techniques in live-cell investigations were presented.

Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

1996 ◽  
Vol 54 ◽  
pp. 730-731
Author(s):  
E. D. Salmon ◽  
J. C. Waters ◽  
C. Waterman-Storer
Keyword(s):  
Imaging System ◽  
Ccd Camera ◽  
Transmitted Light ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Optical Efficiency ◽  
Multiple Band ◽  
Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

α-Methylene-β-Lactone Probe for Measuring Live-Cell Reactions of Small Molecules

2020 ◽  
Author(s):  
Lei Wang ◽  
Louis Riel ◽  
Bekim Bajrami ◽  
Bin Deng ◽  
Amy Howell ◽  
...  
Keyword(s):  
Mass Spectra ◽  
Live Cell ◽  
Live Cells ◽  
The Novel ◽  
Proteomics Analysis ◽  
Chemical Proteomics ◽  
Tandem Mass Spectra ◽  
Modified Peptides ◽  
Covalent Probes ◽  
Ht 29

The novel use of the α-methylene-β-lactone (MeLac) moiety as a warhead of multiple electrophilic sites is reported. In this study, we demonstrate that a MeLac-alkyne is a competent covalent probe and reacts with diverse proteins in live cells. Proteomics analysis of affinity-enriched samples identifies probe-reacted proteins, resolves their modified peptides/residues, and thus characterizes probe-protein reactions. Unique methods are developed to evaluate confidence in the identification of the reacted proteins and modified peptides. Tandem mass spectra of the peptides reveal that MeLac reacts with nucleophilic cysteine, serine, lysine, threonine, and tyrosine residues, through either Michael addition or acyl addition. A peptide-centric proteomics platform, using MeLac-alkyne as the measurement probe, successfully analyzes the Orlistat selectivity in live HT-29 cells. MeLac is a versatile warhead demonstrating enormous potential to expedite the development of covalent probes and inhibitors in interrogating protein (re)activity. MeLac-empowered platforms in chemical proteomics are widely adaptable for measuring the live-cell action of reactive molecules.

The Use of the Vitality and Chemotaxis of Human Polymorphonuclear Leukocytes for the In Vitro Estimation of the Acute Toxicity of the First Ten Chemicals from the MEIC List

1993 ◽  
Vol 21 (1) ◽  
pp. 73-80
Author(s):  
Matteo Valentino ◽  
Francesca Monaco ◽  
Maria Antonietta Pizzichini ◽  
Mario Governa
Keyword(s):  
Ethidium Bromide ◽  
Polymorphonuclear Leukocytes ◽  
Rank Correlation ◽  
Fluorescein Diacetate ◽  
Live Cells ◽  
In Vitro Toxicity ◽  
Ic50 Values ◽  
Acute Cytotoxicity ◽  
Human Polymorphonuclear Leukocytes

The acute cytotoxicity of the first ten MEIC chemicals has been estimated by others in various cell lines. In the present investigation, isolated human polymorphonuclear leukocytes (PMN) from ten healthy non-smoking laboratory personnel were used to assess in vitro toxicity of the same chemicals. The cells were treated with different concentrations of the respective chemicals for three hours and their vitality and chemotaxis were tested. Vitality was measured by fluorescence microscopy after the addition of fluorescein diacetate and ethidium bromide. Living cells which took up and hydrolysed fluorescein diacetate, and dead cells, stained by ethidium bromide, were counted and the percentage of live cells was calculated. Locomotion stimulated by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (F-MLP), was measured in blind-well Boyden chambers and a chemotactic index was calculated. The results were mathematically transformed to produce a linear curve, and then fitted by the linear least squares procedure, from which LC50 and IC50 values were obtained by interpolation. All the chemicals decreased the vitality and inhibited the chemotaxis of the PMN. Obviously the chemotactic test was more sensitive than the vitality one. A correlation (r = 0.933) was found between vitality and chemotaxis inhibition. Spearman rank correlation analysis revealed significant correlations between our results and those from in vitro experiments conducted in other laboratories, as well as with data concerning mouse, rat and human lethal doses.

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