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MethodsX Jun 2024Microcontact printing (MCP) is used to pattern a surface with a specific compound, allowing the spatially restricted response of cells to be assayed as they encounter a...
Microcontact printing (MCP) is used to pattern a surface with a specific compound, allowing the spatially restricted response of cells to be assayed as they encounter a molecule of interest. MCP is a relatively low-cost and accessible technique that uses commercially available reagents and common cell culture equipment. However, it can be technically challenging, slow, and incompatible with microwell cell culture plates that are widely used for screening and other applications. Here, we describe a novel protocol using medical biopsy punches to transfer patterns into standard 96-well plates via polydimethylsiloxane (PDMS) cutouts. We demonstrate that this method can be used to deposit patterns of poly-D-lysine (PDL) into the microwells of glass-bottom plates. As a proof-of-concept, we show that cultured rodent glial cells preferentially grow and extend processes on the pattern. This method will allow larger scale MCP experiments in which different patterns, proteins, or other factors can be assayed in parallel.•Biopsy punches enable both cutting out small circular stamps and plunging them into tissue culture microwells to transfer proteins.•Compared to standard MCP, this method offers a more rapid workflow to pattern proteins onto substrates, and allows use of microwell plates that permits larger-scale experiments.
PubMed: 38524307
DOI: 10.1016/j.mex.2024.102665 -
Pharmaceuticals (Basel, Switzerland) Sep 2023Lorlatinib (LOR) is a third-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor drug. The Food and Drug Administration (FDA) has granted an approval...
Development of Novel Micellar-Enhanced High-Throughput Microwell Spectrofluorimetric Method for Quantification of Lorlatinib: Application to In Vitro Drug Release and Analysis of Urine Samples.
Lorlatinib (LOR) is a third-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor drug. The Food and Drug Administration (FDA) has granted an approval for the use of LOR as a first therapeutic intervention for individuals diagnosed with ALK-positive metastatic and advanced non-small-cell lung cancer (NSCLC). The present study outlines, for the first time, the development and validation of an innovative microwell-based spectrofluorimetric (MW-SFL) method for the quantification of LOR. The proposed method involved the enhancement of the weak native fluorescence of LOR by its micellization into the sodium lauryl sulfate (SLS) micelles. The procedures of the method were conducted in white opaque plates with 96 microwells, and the enhanced fluorescence signals were measured by a fluorescence plate reader at 405 nm after excitation at 310 nm. The measured relative fluorescence intensity (RFI) had a linear relationship with LOR concentrations in the range of 60-1600 ng mL. The limit of detection (LOD) and the limit of quantification (LOQ) were found to be 19 and 56 ng mL, respectively. The method's accuracy and precision were assessed using a recovery study; the recovery values ranged from 99.98% to 101.40%, accompanied by relative standard deviation (RSD) values of 0.42% to 1.59%. The proposed MW-SFL method combined the advantages of the intrinsically high sensitivity of the spectrofluorimetric measurement and the excellent throughput of the microwell-based approach. The results proved the method is effective in the determination of LOR in its pharmaceutical tablets, tablet dissolution testing, as well as in spiked urine with a high degree of precision and accuracy. The MW-SFL method is notable for its simple procedures and utilization of water as a solvent, as well as minimal quantities of sample solutions. These features align with its ecofriendly approach to green chemistry principles. These advantages gave the proposed MW-SFL method a high potential value for the determination of LOR in clinical and quality control laboratories.
PubMed: 37765067
DOI: 10.3390/ph16091260 -
Molecules (Basel, Switzerland) Jul 2023In this study, a new green microwell spectrofluorimetric assay (MW-SFA) with high throughput was developed and validated, for the first time, for the determination of...
Development of a Green Microwell Spectrofluorimetric Assay with High Analytical Throughput for the Determination of Selective Serotonin Reuptake Inhibitors in Pharmaceutical Dosage Forms and Plasma.
In this study, a new green microwell spectrofluorimetric assay (MW-SFA) with high throughput was developed and validated, for the first time, for the determination of three selective serotonin reuptake inhibitors (SSRIs) in pharmaceutical dosage forms and plasma. These SSRIs were fluoxetine (FLX), fluvoxamine (FXM), and paroxetine (PXT), which are commonly prescribed drugs for depression treatment. The MW-SFA is based on the condensation reaction of SSRIs with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) in alkaline media to form highly fluorescent derivatives. The MW-SFA procedures were conducted in 96-microwell white opaque assay plates with a flat bottom and the fluorescence signals were measured using a microplate reader at their maximum excitation and emission wavelengths. The calibration curves were generated with good correlation coefficients (0.9992-0.9995) between the relative fluorescence intensity (RFI) and the SSRI concentrations in the range of 35-800 ng/mL. The limits of detection were in the range of 11-25 ng/mL, and the precision and accuracy were satisfactory. The proposed MW-SFA was successfully applied to the analysis of the SSRIs in their pharmaceutical dosage forms. The statistical analysis for the comparison between the MW-SFA assay results and those of pharmacopeial assays showed no significant differences between the assays in terms of their accuracy and precision. The application of the proposed MW-SFA was extended to successfully analyze SSRIs in plasma samples. The greenness of the assay was confirmed using three different metric tools. The assay was characterized with high throughput properties, enabling the sensitive simultaneous analysis of many samples in a short time. This assay is valuable for rapid routine applications in pharmaceutical quality control units and clinical laboratories for the determination of SSRIs.
Topics: Selective Serotonin Reuptake Inhibitors; Spectrometry, Fluorescence; Fluvoxamine; Plasma; Pharmaceutical Preparations
PubMed: 37446883
DOI: 10.3390/molecules28135221 -
STAR Protocols Sep 2023The cortical organoid is an efficient model for studying human brain neurodevelopment and neurological disease. However, its three-dimensional structure limits real-time...
The cortical organoid is an efficient model for studying human brain neurodevelopment and neurological disease. However, its three-dimensional structure limits real-time observation of internal physiological changes. Here, we present a protocol for an air-liquid interface attachment culture for cortical organoids. We describe steps for transplanting cortical organoid slices and generating the air-liquid interface. We then detail calcium imaging on organoid external neural networks and immunohistochemical staining on confocal plates.
Topics: Humans; Organoids; Brain; Head
PubMed: 37715950
DOI: 10.1016/j.xpro.2023.102502 -
PLoS Biology Mar 2024Cell culture devices, such as microwells and microfluidic chips, are designed to increase the complexity of cell-based models while retaining control over culture...
Cell culture devices, such as microwells and microfluidic chips, are designed to increase the complexity of cell-based models while retaining control over culture conditions and have become indispensable platforms for biological systems modelling. From microtopography, microwells, plating devices, and microfluidic systems to larger constructs such as live imaging chamber slides, a wide variety of culture devices with different geometries have become indispensable in biology laboratories. However, while their application in biological projects is increasing exponentially, due to a combination of the techniques, equipment and tools required for their manufacture, and the expertise necessary, biological and biomedical labs tend more often to rely on already made devices. Indeed, commercially developed devices are available for a variety of applications but are often costly and, importantly, lack the potential for customisation by each individual lab. The last point is quite crucial, as often experiments in wet labs are adapted to whichever design is already available rather than designing and fabricating custom systems that perfectly fit the biological question. This combination of factors still restricts widespread application of microfabricated custom devices in most biological wet labs. Capitalising on recent advances in bioengineering and microfabrication aimed at solving these issues, and taking advantage of low-cost, high-resolution desktop resin 3D printers combined with PDMS soft lithography, we have developed an optimised a low-cost and highly reproducible microfabrication pipeline. This is thought specifically for biomedical and biological wet labs with not prior experience in the field, which will enable them to generate a wide variety of customisable devices for cell culture and tissue engineering in an easy, fast reproducible way for a fraction of the cost of conventional microfabrication or commercial alternatives. This protocol is designed specifically to be a resource for biological labs with limited expertise in those techniques and enables the manufacture of complex devices across the μm to cm scale. We provide a ready-to-go pipeline for the efficient treatment of resin-based 3D-printed constructs for PDMS curing, using a combination of polymerisation steps, washes, and surface treatments. Together with the extensive characterisation of the fabrication pipeline, we show the utilisation of this system to a variety of applications and use cases relevant to biological experiments, ranging from micro topographies for cell alignments to complex multipart hydrogel culturing systems. This methodology can be easily adopted by any wet lab, irrespective of prior expertise or resource availability and will enable the wide adoption of tailored microfabricated devices across many fields of biology.
Topics: Microtechnology; Cell Culture Techniques; Microfluidics; Printing, Three-Dimensional; Lab-On-A-Chip Devices
PubMed: 38478490
DOI: 10.1371/journal.pbio.3002503 -
Journal of the Indian Society of... 2023Antimicrobial intracanal medicaments play a vital role in successful outcome of any endodontic procedure. One such plant extract Cuminium cyminium, as intracanal...
CONTEXT
Antimicrobial intracanal medicaments play a vital role in successful outcome of any endodontic procedure. One such plant extract Cuminium cyminium, as intracanal medicaments needs to be researched.
AIMS
The purpose of this study was in vitro assessment of the antibacterial activity of ethanol extract of C. Cyminium in comparison to Calcium hydroxide (Ca[OH]) as intracanal medicament against the pathogens of endodontic infection, at an interval 1 h, 24 h, 48 h, and 72 h.
SETTINGS AND DESIGN
The study was conducted in the central research laboratory of our institute. Freshly prepared C. cyminium extract was procured from AYUSH approved laboratory and direct contact test (DCT) was utilized, which is based on turbidometric determination of microbial growth in a 96-well microplate, carrying 6 times for each bacteria.
METHODOLOGY
Three groups were assigned for each material in a 96 microwell plate for DCT. Bacterial growth kinetics was monitored at intervals of 1 h, 24 h, 48 h, and 72 h using spectrophotometer at 595 nm. The optical density of T2 (Test group), P2 (Positive control), and N2 (Negative control) was considered.
STATISTICAL ANALYSIS USED
After compiling the data, based on the normality of data, further statistical analysis was conducted using Kolmogorov-Smirnov test, Paired t-test, and pairwise comparisons by Turkey's multiple post hoc procedures. The level of statistical significance was set at P = 0.05.
RESULTS
The comparison of mean optical density values of C. cyminium in comparison with Ca(OH) against the microorganisms of endodontic origin showed a statically significant decrease in bacterial viability at the end of 24 h, 48 h, and 72 h.
CONCLUSION
Based on the results of the study, it can be concluded that C. cyminium has significant antibacterial action against endodontic origin, at interval of 24 h, 48 h, and 72 h.
Topics: Humans; Anti-Infective Agents; Anti-Bacterial Agents; Calcium Hydroxide; Bacteria; Ethanol; Plant Extracts
PubMed: 37861640
DOI: 10.4103/jisppd.jisppd_303_23 -
Biomicrofluidics Jan 2024Discovery of new strains of bacteria that inhibit pathogen growth can facilitate improvements in biocontrol and probiotic strategies. Traditional, plate-based co-culture...
Discovery of new strains of bacteria that inhibit pathogen growth can facilitate improvements in biocontrol and probiotic strategies. Traditional, plate-based co-culture approaches that probe microbial interactions can impede this discovery as these methods are inherently low-throughput, labor-intensive, and qualitative. We report a second-generation, photo-addressable microwell device, developed to iteratively screen interactions between candidate biocontrol agents existing in bacterial strain libraries and pathogens under increasing pathogen pressure. Microwells (0.6 pl volume) provide unique co-culture sites between library strains and pathogens at controlled cellular ratios. During sequential screening iterations, library strains are challenged against increasing numbers of pathogens to quantitatively identify microwells containing strains inhibiting the highest numbers of pathogens. Ring-patterned 365 nm light is then used to ablate a photodegradable hydrogel membrane and sequentially release inhibitory strains from the device for recovery. Pathogen inhibition with each recovered strain is validated, followed by whole genome sequencing. To demonstrate the rapid nature of this approach, the device was used to screen a 293-membered biovar 1 agrobacterial strain library for strains inhibitory to the plant pathogen sp. 15955. One iterative screen revealed nine new inhibitory strains. For comparison, plate-based methods did not uncover any inhibitory strains from the library (n = 30 plates). The novel pathogen-challenge screening mode developed here enables rapid selection and recovery of strains that effectively suppress pathogen growth from bacterial strain libraries, expanding this microwell technology platform toward rapid, cost-effective, and scalable screening for probiotics, biocontrol agents, and inhibitory molecules that can protect against known or emerging pathogens.
PubMed: 38434239
DOI: 10.1063/5.0188270 -
RSC Advances Mar 2024This study describes the prototype of a novel ultra-sensitive time-resolved fluoroimmunoassay (TRFIA) for the quantification of lead (Pb) in plasma. The assay procedures...
A prototype of ultrasensitive time-resolved fluoroimmunoassay for the quantitation of lead in plasma using a fluorescence-enhanced europium chelate label for the detection system.
This study describes the prototype of a novel ultra-sensitive time-resolved fluoroimmunoassay (TRFIA) for the quantification of lead (Pb) in plasma. The assay procedures were conducted in 96-microwell plates and involved the competitive binding format. The assay used a mouse monoclonal antibody, designated as 2C33, that specifically recognized the diethylenetriamine pentaacetic acid chelate of Pb (Pb-DTPA) but did not recognize Pb-free DTPA chelator. The antigen used for coating onto the inner surfaces of assay plate microwells was Pb-DTPA conjugated with bovine serum albumin protein (Pb-DTPA-BSA). The competitive binding reaction occurred between Pb-DTPA chelates, formed in the sample solutions by treating the samples with an excess DTPA, and the coated Pb-DTPA-BSA for a limited quantity of 2C33 antibody binding sites. The antigen-antibody complex formed in the plate wells was quantified by a europium-DTPA-labeled secondary antibody and a fluorescence enhancement solution. The conditions of the assay were refined, and its optimum procedures were established. The TRFIA was validated following the immunoassay validation guidelines, and all of the validation criteria were acceptable. The working range of the assay was 20-300 pg mL and its limit of quantitation was 20 pg mL. Metals that are commonly encountered in blood plasma did not interfere with Pb in the analysis by the proposed TRFIA. The assay was applied to the quantitation of Pb in plasma samples with satisfactory accuracy and precision. The results were compared favorably with those obtained by atomic emission spectroscopy. In conclusion, the present study represents the first TRFIA for the quantitation of Pb in plasma. The assay is superior to the existing atomic spectrometric methods and other immunoassays for Pb in terms of sensitivity, convenience, and analysis throughputs. The proposed TRFIA is anticipated to effectively contribute to assessing Pb concentrations and controlling the exposure of humans to its potential toxicity.
PubMed: 38495999
DOI: 10.1039/d3ra07673c