Multiwell cap assay: A simple objective method for the assessment of leukocyte locomotion in vitro

1986 ◽  
Vol 6 (12) ◽  
pp. 1041-1047 ◽  
Author(s):  
Anne M. Evans ◽  
J. David Chambers ◽  
John R. Hobbs
Keyword(s):  
Objective Method ◽  
Simple Method ◽  
Coefficients Of Variation ◽  
Experimental Protocols ◽  
Micropore Filter

A simple method for evaluating leukocyte locomotion in vitro has been developed and validated for several chemoattractants. The multiwell cap assay (MWCA) comprises chambers constructed from readily available disposable plastics and is quickly assembled, permitting large experimental protocols. Leukocytes which have migrated through a micropore filter are recovered and counted electronically yielding a precise, objective result. Coefficients of variation are approximately 6%.

The Filter Loop Technique as a Method of Measuring Platelet Aggregation in the Flowing Blood of the Rat; the Inhibitory Activity of 5-oxo-l-cyclopentene-l-heptanoic Acid (AY-16,804) on Platelet Aggregation

1973 ◽  
Vol 30 (01) ◽  
pp. 138-147 ◽  
Author(s):  
Christopher R. Muirhead
Keyword(s):  
Platelet Aggregation ◽  
Prostaglandin E1 ◽  
Suitable Model ◽  
Heptanoic Acid ◽  
Simple Method ◽  
Loop Technique ◽  
Flowing Blood ◽  
Filter Loop

SummaryThe filter loop technique which measures platelet aggregation in vivo in the flowing-blood of the rat was compared to the optical density technique of Born which is carried out in vitro with platelet rich plasma. Using these two experimental models the effect on platelet aggregation of three known inhibitors sulfinpyrazone, dipyridamole and prostaglandin E1, and a novel compound 5-oxo-l-cyclopentene-l-heptanoic acid (AY-16, 804) was determined.The effects on platelet aggregation of the known inhibitors were consistent with information in the literature. Prostaglandin E1 was the most potent inhibitor in both techniques; sulfinpyrazone inhibited aggregation in both models but was less potent than prostaglandin E1. AY-16, 804 exhibited activity in vitro and in vivo similar to that of sulfinpyrazone. Dipyridamole did not inhibit platelet aggregation in vivo and did not inhibit aggregation in vitro in concentrations at which it remained soluble.The filter loop technique is a suitable model for measuring platelet aggregation in the flowing blood of the rat. It is a relatively simple method of determining aggregation and easily adapted to other species.

Rhenium-188 labeled recombinant human plasminogen kringle5 (rhk5) and preliminary biodistribution

2011 ◽  
Vol 50 (06) ◽  
pp. 234-239 ◽  
Author(s):  
R. Guo ◽  
Y. Ma ◽  
R. Zhang ◽  
S. Liang ◽  
H. Shen ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Human Serum ◽  
Critical Role ◽  
Angiogenesis Inhibitor ◽  
Simple Method ◽  
Tumour Formation ◽  
Human Plasminogen ◽  
Specificity Of Binding ◽  
A549 Lung

Summary Aim: Angiogenesis plays a critical role in tumour formation and metastasis. Suitable radiolabeled angiogenesis inhibitor can be used for noninvasive imaging of angiogenesis and radionuclide therapy. Here we prepare rhenium-188 labeled recombinant human plasminogen kringle5 (188Re-rhk5) in a convenient manner than evaluate its properties in A549 lung adenocarcinoma. Methods: 188Rerhk5 was obtained by conjugating His group at the C end of rhk5 with fac- [188Re(H2O)3(CO)3]+. Chelating efficiency of fac-[188Re(H2O)3(CO)3]+ and radiolabeling efficiency of 188Re-rhk5 were measured by radio thin-layer chromatography (RTLC). In vitro stability of 188Re-rhk5 was determined in human serum at 37°C and analyzed by RTLC. Competition test was also performed to verify the specificity of binding. A biodistribution study was carried out in nude mice bearing A549 lung adenocarcinoma. Results: 188Rerhk5 was obtained with a radiolabel efficiency of 66.1%, the radiochemical purity (RCP) can marreach 95.2% after purification. 188Re-rhk5 showed high stability in human serum, the RCP was more than 80% even 12 h after incubation. Competition test showed a high binding specificity. Furthermore, this radio-complex was excreted mainly through kidneys and showed specific tumour uptake in mice bearing A549 tumours. Conclusion: 188Re-rhk5 was prepared by a simple method. Preliminary biodistribution results showed its potential as an agent for possible tumour imaging, therapy and encouraged further investigation.

Morphometric Registration of Cytotoxic Changes of Epithelial Cells In Vitro: A Methodological Study

1990 ◽  
Vol 17 (3) ◽  
pp. 174-176
Author(s):  
Lis Andersen ◽  
Dorthe Arenholt-Bindslev
Keyword(s):  
Culture System ◽  
Optimal Allocation ◽  
Volume Density ◽  
Cell Culture System ◽  
Allocation Of Resources ◽  
Methodological Study ◽  
Epithelial Cell Culture ◽  
Coefficients Of Variation ◽  
And Control

Quantification of toxicity-induced cytomorphological effects in an epithelial cell culture system is described. Estimates of volume density and star volume of mitochondria and lysosomes are given. Mean volumes (n = 5) and coefficients of variation of these parameters were equal in experimental (TPA-treatment) and control cultures. An optimal allocation of resources for estimating cytomorphometric parameters would be to increase the number of culture flasks.

scholarly journals Cemp1-p3 Peptide Promotes the Transformation of Octacalcium Phosphate into Hydroxyapatite Crystals

Crystals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1131
Author(s):  
Maricela Santana ◽  
Gonzalo Montoya ◽  
Raúl Herrera ◽  
Lía Hoz ◽  
Enrique Romo ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Octacalcium Phosphate ◽  
Sequence Motifs ◽  
Simple Method ◽  
Transmission Electron ◽  
Precursor Phase ◽  
Mineralized Tissues ◽  
Potent Growth

Dental cementum contains unique molecules that regulate the mineralization process in vitro and in vivo, such as cementum protein 1 (CEMP1). This protein possesses amino acid sequence motifs like the human recombinant CEMP1 with biological activity. This novel cementum protein 1-derived peptide (CEMP1-p3, from the CEMP1’s N-terminal domain: (QPLPKGCAAVKAEVGIPAPH), consists of 20 amino acids. Hydroxyapatite (HA) crystals could be obtained through the combination of the amorphous precursor phase and macromolecules such as proteins and peptides. We used a simple method to synthesize peptide/hydroxyapatite nanocomposites using OCP and CEMP1-p3. The characterization of the crystals through scanning electron microscopy (SEM), powder X-ray diffraction (XRD), high--resolution transmission electron microscopy (HRTEM), and Raman spectroscopy revealed that CEMP1-p3 transformed OCP into hydroxyapatite (HA) under constant ionic strength and in a buffered solution. CEMP1-p3 binds and highly adsorbs to OCP and is a potent growth stimulator of OCP crystals. CEMP1-p3 fosters the transformation of OCP into HA crystals with crystalline planes (300) and (004) that correspond to the cell of hexagonal HA. Octacalcium phosphate crystals treated with CEMP1-p3 grown in simulated physiological buffer acquired hexagonal arrangement corresponding to HA. These findings provide new insights into the potential application of CEMP1-p3 on possible biomimetic approaches to generate materials for the repair and regeneration of mineralized tissues, or restorative materials in the orthopedic field.

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.

Coating of Polystyrene Surface with REDV-Gelatin Conjugate for Selective Isolation of Endothelial Cells

2021 ◽  
Vol 904 ◽  
pp. 287-292
Author(s):  
Wan Song Zhang ◽  
Ya Jie Fu ◽  
Rui Wang ◽  
Xuan Hui Qiu ◽  
Ze Yuan Feng ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular Function ◽  
Flow Cytometric Analysis ◽  
Internal Surface ◽  
Cost Effective ◽  
Cell Viability Assay ◽  
Simple Method ◽  
Migration Assay ◽  
Gelatin Coating

Endothelial cells (EC), which line the internal surface of blood vessels, play various essential roles in controlling vascular function. The mouse is an important animal model for the study of vascular biology and cardiovascular diseases. However, the isolation of primary EC from the murine aorta is challenging because they are readily contaminated by smooth muscle cells (SMC). A previous study developed a simple method to isolate murine EC from SMC. By taking advantage of the differential sedimentation rate between the two cells, the EC was selectively enriched with collagen-coated polystyrene surfaces. Our study further improved this method by introducing a biomimetic peptide REDV (Arg-Glu-Asp-Val), which may bind specifically to EC but not to SMC or fibroblasts. Firstly, REDV-gelatin conjugate was synthesized by using the amine-to-sulfhydryl crosslinker SMCC. REDV-gelatin coating was then prepared on polystyrene surfaces, and their affinities to EC and SMC were subsequently investigated. Fluorescence microscopy and flow cytometric analysis showed that EC adhesion to the gelatin coating was significantly promoted by REDV peptide conjugation. Moreover, cell migration assay and cell viability assay also showed that the conjugation of REDV does not affect EC migration, and this coating did not show cytotoxicity against EC. This gelatin-REDV coating provides a cost-effective and straightforward tool for isolating EC from SMC, which may facilitate in vitro investigations of EC from mice.

In Vitro Evaluation of Capturing and Dissolving the Clot by Magnetic Nanoparticles Carrying a Thrombolytic Agents Instead of Temporary Inferior Vena Cava (IVC) Filter

2020 ◽  
Vol 16 (11) ◽  
pp. 1623-1632
Author(s):  
Abbas Moghanizadeh ◽  
Fakhreddin Ashrafizadeh ◽  
Jaleh Varshousaz ◽  
Mahshid Kharaziha
Keyword(s):  
Magnetic Field ◽  
Magnetic Nanoparticles ◽  
Vena Cava ◽  
Simple Method ◽  
Thrombolytic Agents ◽  
Blood Clots ◽  
Life Threatening ◽  
The Magnetic Field ◽  
Different Levels

This study aims to evaluate the efficiency of a novel in vitro technique in clot capturing and dissolving them by applying magnetic force on magnetic nanoparticles (MNP) carrying thrombolytic agents. It is a quick and simple method to protect patients from a life-threatening pulmonary embolism in an emergency to provide time for the medical team. To analyze the in vitro efficiency of nano-magnetic capturing and dissolving of clots (NCDC), different levels of process parameter including strength magnetic field (0.1, 0.2 and 0.3 T) and fluid flow rate (2.5, 5 and 7 l/min) are exposed to different blood clots sizes from 5 × 10 to 20 × 10 mm2 (length × diameter), in an in vitro flow model. The results show that by increasing the parameters to their maximum values, it is possible to immobilize 100% of the clots and dissolve around 61.4% of clots weight. In addition, the clot-dissolving is directly proportional to the magnetic field strength. NCDC is an efficient technique in immobilizing and dissolving the clots and its efficiency depends on process parameters especially the magnetic field.

scholarly journals A simple method of suspending implant wear particles for more physiological modelling of cell-particle interactions in vitro

2021 ◽  
Author(s):  
Christine Poon
Keyword(s):  
Wear Debris ◽  
Culture Media ◽  
Polymeric Materials ◽  
Wear Particle ◽  
Physicochemical Characteristics ◽  
Wear Particles ◽  
Simple Method ◽  
Implant Wear

AbstractArthroplasty implants e.g. hip, knee, spinal disc sustain relatively high compressive loading and friction wear, which lead to the formation of wear particles or debris between articulating surfaces. Despite advances in orthopaedic materials and surface treatments, the production of wear debris from any part of a joint arthroplasty implant is currently unavoidable. Implant wear debris induces host immune responses and inflammation, which causes patient pain and ultimately implant failure through progressive inflammation-mediated osteolysis and implant loosening, where the severity and rate of periprosthetic osteolysis depends on the material and physicochemical characteristics of the wear particles. Evaluating the cytotoxicity of implant wear particles is important for regulatory approved clinical application of arthroplasty implants, as is the study of cell-particle response pathways. However, the wear particles of polymeric materials commonly used for arthroplasty implants tend to float when placed in culture media, which limits their contact with cell cultures. This study reports a simple means of suspending wear particles in liquid medium using sodium carboxymethyl cellulose (NaCMC) to provide a more realistic proxy of the interaction between cells and tissues to wear particles in vivo, which are free-floating in synovial fluid within the joint cavity. Low concentrations of NaCMC dissolved in culture medium were found to be effective for suspending polymeric wear particles. Such suspensions may be used as more physiologically-relevant means for testing cellular responses to implant wear debris, as well as studying the combinative effects of shear and wear particle abrasion on cells in a dynamic culture environments such as perfused tissue-on-chip devices.

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