Glass Bottom Dishes Research Articles

8660 Bodipy Labeled Vitamin D3 Associates With Lipid Droplets In Adipocytes

Abstract Disclosure: N. Ucar: None. J.T. Deeney: None. R. Pickering, PhD: None. M.T. Kirber: None. T. Fan: None. R. Loo, PhD: Research Investigator; Self; Carbogen Amcis BV. M.F. Holick: Grant Recipient; Self; Carbogen Amcis BV. Research Investigator; Self; Carbogen Amcis BV. Obese patients are often found to be deficient in vitamin D, requiring higher levels of supplementation than normal weight subjects. While this has been attributed to vitamin D3‘s lipid solubility and accumulation in fat tissue, little is known about how vitamin D3 enters adipocytes and associates with the intracellular lipid droplet. The goal of this study was to investigate this association using a novel fluorescently labelled vitamin D3. Bodipy, 4,4-difluoro-1,3,5, 7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503), was covalently linked to vitamin D3 and its structure was confirmed by NMR and mass spectroscopy. Murine 3T3L1 and primary adipocytes were used for our study. Mouse 3T3L1 preadipocytes were cultured in multiwell plates or glass bottom dishes in high glucose DMEM media and differentiated for 7-14 days. Primary adipocytes were obtained by collagenase digestion after surgical extraction of adipose tissue from male/female C57BL/6J mice and attached to glass bottom dishes. The cultured adipocytes were exposed to 10 microM BODIPY labelled Vitamin D3 (vitD-B). BODIPY was added to cultured cells and served as the control. Cells were imaged at various times on a wide-field epifluorescence microscope and analysed with NIH ImageJ software. VitD-B adipocyte uptake and accumulation in lipid droplets was observed over 24 hrs. Isolated lipid droplets were also incubated with vitD-B and BODIPY. VitD-B in mature 3T3L1 cells was observed in lipid droplets after 1h incubation and increased fluorescent intensity was observed over the next 24 hrs. BODIPY was observed in the lipid droplets after 15-20 mins. As a negative control, non-differentiated 3T3L1 cells did not exhibit staining by BODIPY even after 24-hrs. When vitD-B was added to primary cultured adipocytes, it appeared in the adipocytes within 1-hr. Fluorescence intensity of primary adipocytes was increased 1.2-fold at 8 hrs and 5.7-fold at 24 hrs compared to 1 hr. For comparison the positive control treated with BODIPY exhibited rapid fluorescence uptake into primary adipocytes that was increased 29-fold higher than that of vitD-B at 1 hr. VitD-B and BODIPY both demonstrated rapid uptake into isolated lipid droplets (less than 2 min) based on fluorescence microscopy.These results hold promising implications for understanding of the interaction between vitamin D and intracellular lipid droplets. Keywords: Adipocyte, Vitamin D3, BODIPY, adipocytes, lipid droplet, fluorescent labelled vitamin D3 Presentation: 6/2/2024

A hyaluronic acid-containing reagent compatible with glass-bottom dishes and capable of sustained binding of human spermatozoa.

Commercial products currently available for sperm selection utilizing hyaluronic acid (HA) binding prior to intracytoplasmic sperm injection (ICSI) are widely used but have some disadvantages. To potentially circumvent these limitations, we compared ICSI using a self-made hyaluronic acid (smHA) reagent with ICSI using SpermSlow. The binding of the reagents to spermatozoa on plastic- or glass-bottom dishes was quantitated using spermatozoa that were isolated by density-gradient centrifugation and swim-up procedures (N=10/group). Additionally, we investigated the relationship between the HA reagent used prior to ICSI and clinical outcomes after assisted reproduction with HA-ICSI (N=81). The smHA reagent exhibited extremely stable binding to human spermatozoa. The binding time of spermatozoa was significantly longer in the smHA reagent than in SpermSlow on both plastic and glass dishes (plastic: 60.0±0.0 min vs. 2.7±5.9 min, P<0.001; glass: 60.0±0.0 min vs. 2.5±1.8 min, P<0.001). There were no significant differences in the normal fertilization rate between HA-ICSI with the smHA reagent (128/160, 80.0%) and HA-ICSI with SpermSlow (171/231, 74.0%, P=0.184). The frequency of the blastocyst development from the HA-ICSI-derived zygote was significantly higher with the smHA reagent (74/101, 73.3%) than with SpermSlow (76/131, 58.0%, P=0.019). The rates of biochemical pregnancy, clinical pregnancy, fetal heart movement, live birth, and miscarriage were not significantly different between HA-ICSI with the smHA reagent and HA-ICSI with SpermSlow. The blastulation rate was higher for HA-ICSI with the smHA reagent as compared with SpermSlow. Clinical outcomes, excluding blastulation, after HA-ICSI were the same using smHA reagent and using SpermSlow. Spermatozoa binding to the smHA reagent was not attenuated over a 60-min time course. In conclusion, this reagent may shorten and simplify HA-ICSI procedures because smHA can be used with any dish material, making it easier to observe the spindle or assess intracytoplasmic morphology.

Functional profiling of resident cells during vascular aging

Introduction: Depending on surrounding cell types, substrate mechanics, and chemical agonists, vascular cell types – including endothelial (ECs) and smooth muscle cells (VSMC) — modulate their behavior to maintain homeostasis. Aging perturbs this homeostasis resulting in EC and SMC dysfunction, marked by impaired barrier integrity and pathological ECM remodeling. Endothelial-to-mesenchymal transition (EndMT) in ECs and “dedifferentiation” of VSMCs towards a synthetic phenotype are phenomenon under investigation as cellular drivers of the widespread dysregulation seen with aging. The overall goal of this project is to understand the effect of cross-talk between ECM and growth factor signaling on vascular cell plasticity during aging. Materials and Methods: Human aortic endothelial cells (HAECs) were seeded onto transwell inserts after seven days treatment with or without TGF-β. After growing to confluence on the insert, cells were subjected to a FITC-dextran transwell assay. A parallel group of cells was seeded onto fibronectin coated glass-bottom dishes and stained for the expression of focal adhesion proteins and cell-type markers. Mouse aortic smooth muscle cells (maSMCs) were isolated from C57Bl6 mice at 3 (young), 6 (early middle-aged; EMA), 9 (middle-age; MA), or greater than 18 months of age (old). Cells were seeded onto dye-quenched (DQ) collagen type I (Col-I) substrate before gain of fluorescence was measured in proximity to the cell as well as in the surrounding media. A parallel group of cells were allowed to grow in media containing FITC Col-I, and gain of fluorescence was measured in proximity to the cells. Each cell type (3M, 6M, 9M, and 18M) in both experiments (DQ and FITC) were further treated with MMP inhibitor, TG2 inhibitor and TGF-β1 to measure their impacts on Col-I turnover. Results: Barrier function was not impaired in ECs cultured with TGF-β1, relative to their untreated counterparts. Mouse aortic SMCs isolated from older mice had decreased rates of Col-I cleavage and were more susceptible to MMP inhibition than their younger counterparts. Conclusions: maSMCs from older mice have lower Col-I turnover than their younger counterparts. This is consistent with bulk tissue behavior, as it is well known that relative collagen levels increase in the elastic vasculature as we age. TGF-β1 treatment did not have an effect on permeability of 4kd FITC-dextran, it is feasible that there will be an impact on the larger 70kd fragments. Travis Brady is supported by the NASEM Ford Foundation Pre-doctoral Fellowship. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Heparan Sulfate Modulation Affects Breast Cancer Cell Adhesion and Transmigration across In Vitro Blood-Brain Barrier.

The disruption of endothelial heparan sulfate (HS) is an early event in tumor cell metastasis across vascular barriers, and the reinforcement of endothelial HS reduces tumor cell adhesion to endothelium. Our recent study showed that while vascular endothelial growth factor (VEGF) greatly reduces HS at an in vitro blood-brain barrier (BBB) formed by human cerebral microvascular endothelial cells (hCMECs), it significantly enhances HS on a breast cancer cell, MDA-MB-231 (MB231). Here, we tested that this differential effect of VEGF on the HS favors MB231 adhesion and transmigration. We also tested if agents that enhance endothelial HS may affect the HS of MB231 and reduce its adhesion and transmigration. To test these hypotheses, we generated an in vitro BBB by culturing hCMECs on either a glass-bottom dish or a Transwell filter. We first quantified the HS of the BBB and MB231 after treatment with VEGF and endothelial HS-enhancing agents and then quantified the adhesion and transmigration of MB231 across the BBB after pretreatment with these agents. Our results demonstrated that the reduced/enhanced BBB HS and enhanced/reduced MB231 HS increase/decrease MB231 adhesion to and transmigration across the BBB. Our findings suggest a therapeutic intervention by targeting the HS-mediated breast cancer brain metastasis.

Viscoelastic relaxation of fibroblasts over stiff polyacrylamide gels by atomic force microscopy

Cell viscoelasticity provides mechanistic insights into fundamental biological functions and may be used in many applications. Using atomic force microscopy in time and frequency domains, we find a peculiar behavior in the viscoelastic relaxation of L929 mouse fibroblasts that may help understand how cells perceive and adapt to distinct extracellular environments. They are stiffer when cultured over polyacrylamide gels (20-350 kPa) than over glass-bottom Petri dishes. The stiffness enhancement of cells over gels is attributed to a significant increase in the low-frequency storage shear moduli compared to the loss moduli, indicating that gels induce a remodeling of cytoskeleton components that store elastic energy. Morphological alterations are then expressed by the fractal dimension measured on confocal images of the f-actin cytoskeleton. We show a direct scaling between the fractal dimension and the substrate’s rigidity.

A low-cost alternative method of generating fibronectin micropatterned lines for cellular applications

The cellular microenvironment contributes to the architecture, differentiation, polarity, mechanics and functions of the cell [1]. Spatial confinement of cells using micropatterning techniques allows to alter and regulate the cellular microenvironment for a better understanding of cellular mechanisms [2]. However, commercially available micropatterned consumables such as coverslips, dishes, plates etc. are expensive.These methods are complex and based on deep UV patterning [3,4]. In this study, we establish a low-cost method for effective micropatterning using Polydimethylsiloxane (PDMS) chips.•We demonstrate this method by generating fibronectin-coated micropatterned lines (width, 5 µm) on a glass bottom dish.•As a proof of concept, we culture macrophages on these lines. We additionally show that this method allows to determine the cellular polarity by measuring the position of the nucleus within a cell on a micropatterned line.

Light-Induced Condensation of Biofunctional Molecules around Targeted Living Cells to Accelerate Cytosolic Delivery.

The light-induced force and convection can be enhanced by the collective effect of electrons (superradiance and red shift) in high-density metallic nanoparticles, leading to macroscopic assembly of target molecules. We here demonstrate application of the light-induced assembly for drug delivery system with enhancement of cell membrane accumulation and penetration of biofunctional molecules including cell-penetrating peptides (CPPs) with superradiance-mediated photothermal convection. For induction of photothermal assembly around targeted living cells in cell culture medium, infrared continuous-wave laser light was focused onto high-density gold-particle-bound glass bottom dishes exhibiting plasmonic superradiance or thin gold-film-coated glass bottom dishes. In this system, the biofunctional molecules can be concentrated around the targeted living cells and internalized into them only by 100 s laser irradiation. Using this simple approach, we successfully achieved enhanced cytosolic release of the CPPs and apoptosis induction using a pro-apoptotic domain with a very low peptide concentration (nM level) by light-induced condensation.

Abstract P3043: Functional Role Of Endothelial Mitochondrial Oxidative Stress In Cognitive Impairment: Effects Of L-arginine In Hypertensive Frail Older Adults

Background: Hypertension is common in older adults, particularly in frailty. The association of hypertension and frailty leads to a high-risk of cognitive impairment in older adults. Endothelial dysfunction has been shown to underlie both hypertension and cognitive impairment. L-Arginine is an amino acid involved in many biological processes and is a substrate of two enzymes: nitric oxide (NO) synthase (NOS) and arginase (NOA). L-Arginine is fundamental for NO production by endothelial cells, regulating vascular tone and cardiovascular homeostasis, improving inflammation and oxidative stress. On these grounds, we hypothesized that L-Arginine could improve cognitive impairment via its effect on endothelial mitochondria. Methods: To assess the effects of L-Arginine on endothelial oxidative stress and cognitive function, we used an in vitro and a clinical approach. First, we treated with L-Arginine human umbilical vein endothelial cells (HUVECs). HUVECs were cultured in EGM-2 medium and incubated at 37°C and 5% CO 2 . HUVECs were plated on glass bottom culture dishes and treated with Angiotensin II (1 μM) and Angiotensin II with L-Arginine (500 μM) in EGM-2 medium for 24h. The generation of reactive oxygen species (ROS) was quantified using MitoSOX™ Red (an established ROS probe that accumulates in mitochondria), incubating cells for 10 min at 37°C and 5% CO 2 , as we previously described. We then designed a single-blind, placebo-controlled, parallel-group, randomized trial to observe the effects of 4-weeks oral supplementation of L-Arginine on global cognitive function of hypertensive frail older patients. Results: In vitro , we demonstrated that L-Arginine significantly attenuated Angiotensin II-induced oxidative stress in endothelial cells. Co-treatment of HUVECs with Ang II and L-Arginine significantly attenuated mitochondrial ROS production (p&lt;0.001). In the clinical study, we evaluated 35 frail hypertensive elderly patients assigned to L-Arginine and 37 assigned to placebo: at follow-up, we found a significantly improved Montreal Cognitive Assessment (MoCA) test score in the L-Arginine treated arm compared to placebo (p&lt;0.05). Conclusions: Taken together, our findings indicate for the first time that oral L-Arginine supplementation improves cognitive impairment in frail hypertensive older adults through a mechanism that involves endothelial mitochondrial ROS.

Abstract 3314: Glioblastoma treatment using epigenetic modification induced by microsecond pulsed electric field (µsPEF) exposure

Abstract Epigenetic modifications derived from changes in sub-cellular localization and activity of the post-translational histone modifying enzymes, histone deacetylases (HDACs) are one of the strategies to treat various types of cancer. Aberrant HDAC activity is an indicator of glioblastoma multiforme (GBM), generally leading to hypoacetylation of histones and a transcriptionally repressed chromatin state. Most noticeably, HDAC4 expression increases 61,000% in brain tumors!1 Upregulation of HDAC4 in human glioma cells (U87-MG) stimulates proliferation and invasiveness, making HDAC4 a therapeutic target. The effectiveness of pharmacological HDAC inhibitors is hampered by non-specific targeting and low selectivity, especially in solid tumors like GBM, and by side-effects with systemic delivery, e.g. cardiac toxicity.2 Existing electroporation-based approaches to GBM aim to induce cell death either directly3 or in combination with adjuvants such as excess extracellular calcium4 or chemotherapy drugs5.A cytostatic approach is introduced in which the initial electroporation from non-thermal, localized µsPEF exposure elicits downstream epigenetic responses that decrease cell proliferation, circumventing obstacles experienced by pharmacological agents. Accumulation of HDAC4 in the nucleus of U87-MG cells following exposure to µsPEF is hypothesized to lead in a dose-dependent manner to cell death via apoptosis and decreased proliferation.A BTX GeminiX2 electroporator delivered square-wave µsPEF of 100 µs and 1.45 kV/cm, while the number of pulses (0, 1, 10, 20 pulses, delivered at 1 Hz) were varied to determine thresholds for HDAC4 translocation and proliferation reduction of U87-MG cells. For immunofluorescence assay to track HDAC4 location at 3 h after exposure, cells were cultured and exposed on glass-bottom dishes. For MTT and ATP kinase assays, cells were exposed in electroporation cuvettes and transferred to 96-well plates to measure proliferation at 6, 24 and 72 h after exposure. The ratio of HDAC4 in the nucleus compared to the cytoplasm (N/C ratio) more than doubles after 20 pulses, compared to sham control with no µsPEF exposure. Cell concentration relative to control drops over 90% given 20 pulses. Our data reflect the inhibition of cell proliferation and HDAC4 accumulation in nuclei. Although electroporation-based therapies for GBM have been studied pre-clinically, this study delves further into fundamental mechanisms and optimization of the energy delivered by µsPEF exposure that induces cell death with respect to epigenetic modification. 1. Lee P. et al. Anticancer Res, 35:615, 2015.2. Slingerland M. et al. Anticancer Drugs, 25:140, 2014.3. Rossmeisl J.H. et al. J Neurosurg, 123:1008, 2015.4. Wasson E.M. et al. Ann Biomed Eng, 45:2535, 2017.5. Sharabi S. et al. Sci Rep, 10:2178, 2020. Citation Format: Zahra Safaei, Gary L Thompson. Glioblastoma treatment using epigenetic modification induced by microsecond pulsed electric field (µsPEF) exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3314.

Enhanced Isolation of Single Myofibers in Flexor Digitorum Brevis Dissociation

IntroductionThe isolation of single myofibers from the Flexor Digitorum Brevis (FBD) has been a well‐established model in skeletal muscle physiology research. The use of isolated single‐fibers has led to advancements in the understating of the excitation – contraction (E‐C) coupling mechanisms in skeletal muscle, calcium transients, the role of mitochondria, and much more. However, these experiments rely on the quality of the dissociation of myofibers and their attachment to the imaging dish which can be difficult and time exhaustive. The purpose of this study was to build upon previously established methodologies to improve the yield of intact‐alive myofibers in a time efficient manner.Methods and MaterialsEuthanasia and experimental protocols were approved by the University of Texas at Arlington’s IACUC. Based upon previously established dissociation techniques (1–3), we modified the protocol as follows: the intact FDB was dissected out and placed in 2 mL of Ringer’s solution (142 mM NaCl2, 5 mM KCl, 1.8 mM MgCl2, 10 mM Hepes, 10 mM Glucose). Under the dissection microscope the whole muscle was separated into 4 muscle bundles; separated by the distal tendons. The muscle bundles were transferred to the digestion media (DMEM with glutamine, 2% sterile filtered fetal bovine serum (FBS), 0.1% gentamycin, and 4 mg ml‐1 Collagenase A). The muscle bundles were incubated for 30‐45 minutes at 37° C in 5% CO2. Following incubation, the bundles were transferred to 1mL of Ringer’s solution in a 24‐well plate and gently titrated with the cut end of a P1000 pipette tip. The solution was transferred to laminin coated glass bottom dishes and incubated for 15‐30 minutes for fiber attachment. Following fiber attachment, the fibers were either stained with the LIVE/DEAD™ Viability/Cytoxicity kit or loaded with Fura‐2 AM fluorescent dye for measurement of maximal calcium depolarization response induced with 100mM Potassium Chloride (KCl) using a PTI spectrophotometry system.Results and ConclusionWe found that using an increased concentration of collagenase and a reduced time of digestion successfully increased the number of fibers in the field of vision.. Laminin coated glass bottom dishes produced quick and strong attachment, allowing for optimal stimulation conditions. The use of cell culture media with FBS protected the sarcolemma increasing the viability of the fibers. This is shown in both the LIVE/DEAD imaging and the response to KCl stimulation. This study demonstrates that the change of dissociation media and the decrease in dissociation time allow for protection of the myofiber for an increased yield and viability. This protocol will allow for improved experimental outcomes in which mature myofibers are subjected to electrical or chemical stimulations.

Temperature and Tumor Microenvironment Dual Responsive Mesoporous Magnetic Nanospheres for Magnetothermal Effect-Induced Cancer Theranostics

Temperature and Tumor Microenvironment Dual Responsive Mesoporous Magnetic Nanospheres for Magnetothermal Effect-Induced Cancer Theranostics

Live Imaging of Axonal Transport in the Adult Drosophila Central Nervous System.

Live imaging of axons allows for the determination of motility and directionality of proteins or organelles. In Drosophila, axonal transport has been predominantly characterized in peripheral neurons, such as larval motor neurons and sensory neurons of the adult wing. As peripheral neurons and central nervous system (CNS) neurons are inherently different, we provide a method to live-image axonal transport of CNS neurons in the cervical connective using an upright or inverted microscope. The method involves dissecting and mounting an entire CNS in a glass bottom petri dish, which is suitablefor imagingofnearly any axon in cervical connective. Here, we show an example for simultaneous imaging of both giant fiber axons, which are part of the fly's escape response circuitry, and due to their large diameter provide outstanding resolution.

Collagen Sandwich Culture of Primary Hepatocytes for Image-Based Investigations.

Collagen sandwich culture of hepatocytes retains many in vivo-like properties and is used for many investigations in liver cell biology, and hepatic pharmacology and toxicology. This chapter describes the method of establishing collagen sandwich culture of hepatocytes in a glass bottom dish for image-based studies.

Inhibition of Carbonic Anhydrase IX Suppresses Breast Cancer Cell Motility at the Single-Cell Level.

Protein Carbonic Anhydrase IX (CA IX), which is expressed in various hypoxic solid tumors in order to maintain proper pH, is also related to cancer cell adhesion, invasion, and metastasis processes. Here, we investigated whether CA IX inhibition by a highly CA IX selective agent benzenesulfonamide VD11-4-2 triggers changes in individual cell motility. We seeded breast cancer cells on an extracellular matrix-coated glass-bottomed dish and in a microfluidic device with a gradient flow of epidermal growth factor (EGF), tracked individual cell movement, calculated their migration speeds, and/or followed movement direction. Our results showed that the inhibitor VD11-4-2 decreased the speed of CA IX positive breast cancer cells by 20–26% while not affecting non-cancerous cell migration. The inhibitor suppressed the cell migration velocity increment and hindered cells from reaching their maximum speed. VD11-4-2 also reduced CA IX, expressing cell movement towards the growth factor as a chemoattractant. Such a single cell-based migration assay enabled the comprehensive investigation of the cell motility and revealed that VD11-4-2 shows the ability to suppress breast cancer cell migration at a lower concentration than previously tested CA IX inhibitors.

An Aggregation-Induced Emission Optical Highlighter for the Studies of Endoplasmic Reticulum-Lipid Droplet Content Dynamics

An Aggregation-Induced Emission Optical Highlighter for the Studies of Endoplasmic Reticulum-Lipid Droplet Content Dynamics

An Integration Strategy to Develop Dual-State Luminophores with Tunable Spectra, Large Stokes Shift, and Activatable Fluorescence for High-Contrast Imaging

An Integration Strategy to Develop Dual-State Luminophores with Tunable Spectra, Large Stokes Shift, and Activatable Fluorescence for High-Contrast Imaging

Resolvins D3, D4, and D5 activate selective signaling pathways to increase intracellular [Ca 2+ ] in rat conjunctival goblet cells

Purpose Resolvins (Rvs) D3, D4, and D5 are specialized proresolving mediators synthesized from the w3 fatty acid docosahexaenoic acid. The purpose of this study was to determine if RvD3, RvD4, or RvD5 interacts with rat conjunctival goblet cells and determine the signal transduction pathways utilized to increase intracellular [Ca2+] ([Ca2+]i). Methods Conjunctiva from male Sprague-Dawley rats was removed and placed in organ culture with RPMI media. Goblet cells were allowed to grow out of the tissue plug. First passage cells were used for all experiments. To measure [Ca2+]i, cells were trypsinized and transferred to 35-mm glass bottom dishes. The cells were incubated at 37°C for 1 hour with the calcium indicator fura2/AM with 250 mM sulfinpyrazone and 8 mM Pluronic acid F127. [Ca2+]i was measured with a ratio imaging system (InCytIm2; Intracellular Imaging, Cincinnati, OH) using wavelengths of 340 and 380 nm and an emission wavelength of 505 nm. Cells were stimulated with RvD3, RvD4, and RvD5 from 10-12-10-8 M. Inhibitors were added 10-30 minutes prior to stimulation. Results RvD3, RvD4, and RvD5 each increased [Ca2+]i in a concentration dependent manner with peak concentrations of 10-9 M for each resolvin. Only RvD5-stimulated increase in [Ca2+]i was inhibited by BOC2, an inhibitor or the ALX/FPR2 receptor. [Ca2+]i stimulated by all three D-series resolvins was blocked by inhibition of phospholipase (PL) C with U73122___ and D with tert 1- butanol, but not A2 with aristolochic acid. Absence of extracellular Ca2+ also blocked the increase in [Ca2+]i stimulated by RvD3, RvD4, and RvD5. Only RvD3-stimulated increase in [Ca2+]i was blocked by H89, an inhibitor of cAMP dependent-protein kinase A. Conclusion RvD3, RvD4, and RvD5, in addition to RvD1 and RvD2, each play a role in the physiologic regulation of conjunctival goblet cells, but use selective intracellular signaling pathways.

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy.

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Building Block Symmetry Relegation Induces Mesopore and Abundant Open-Metal Sites in Metal–Organic Frameworks for Cancer Therapy

Building Block Symmetry Relegation Induces Mesopore and Abundant Open-Metal Sites in Metal–Organic Frameworks for Cancer Therapy

Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model.

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major clinical challenge because no efficient method exist for their elimination once their dissemination is underway. A better understanding of the mechanisms regulating cancer cell invasion may lead to the development of novel potent therapies. Due to their physiological resemblance to tumors, spheroids embedded in collagen I have been extensively utilized by researchers to study the mechanisms governing cancer cell invasion into the extracellular matrix (ECM). However, this assay is limited by (1) a lack of control over the embedding of spheroids into the ECM; (2) high cost of collagen I and glass bottom dishes, (3) unreliable immunofluorescent labeling, due to the inefficient penetration of antibodies and fluorescent dyes and (4) time-consuming image processing and quantification of the data. To address these challenges, we optimized the three-dimensional (3D) spheroid protocol to image fluorescently labeled cancer cells embedded in collagen I, either using time-lapse videos or longitudinal imaging, and analyze cancer cell invasion. First, we describe the fabrication of a spheroid imaging device (SID) to embed spheroids reliably and in a minimal collagen I volume, reducing the assay cost. Next, we delineate the steps for robust fluorescence labeling of live and fixed spheroids. Finally, we offer an easy-to-use Fiji macro for image processing and data quantification. Altogether, this simple methodology provides a reliable and affordable platform to monitor cancer cell invasion in collagen I. Furthermore, this protocol can be easily modified to fit the users' needs.