共查询到20条相似文献,搜索用时 15 毫秒
1.
Marta Wo
niak Sebastian Makuch Gabriela Pastuch-Gawoek Jerzy Winiewski Wiesaw Szeja Martyna Nowak Monika Krawczyk Siddarth Agrawal 《Molecules (Basel, Switzerland)》2021,26(9)
Patients with hematologic malignancies require intensive therapies, including high-dose chemotherapy. Antimetabolite–methotrexate (MTX) has been used for many years in the treatment of leukemia and in lymphoma patients. However, the lack of MTX specificity causes a significant risk of morbidity, mortality, and severe side effects that impairs the quality of patients’ life. Therefore, novel targeted therapies based on the malignant cells’ common traits have become an essential treatment strategy. Glucose transporters have been found to be overexpressed in neoplastic cells, including hematologic malignancies. In this study, we biologically evaluated a novel glucose–methotrexate conjugate (Glu–MTX) in comparison to a free MTX. The research aimed to assess the effectiveness of Glu–MTX on chosen human lymphoma and leukemia cell lines. Cell cytotoxicity was verified by MTT viability test and flow cytometry. Moreover, the cell cycle and cellular uptake of Glu–MTX were evaluated. Our study reveals that conjugation of methotrexate with glucose significantly increases drug uptake and results in similar cytotoxicity of the synthesized compound. Although the finding has been confined to in vitro studies, our observations shed light on a potential therapeutic approach that increases the selectivity of chemotherapeutics and can improve leukemia and lymphoma patients’ outcomes. 相似文献
2.
Zizheng Shen Hansen Zhao Huan Yao Xingyu Pan Jinlei Yang Sichun Zhang Guojun Han Xinrong Zhang 《Chemical science》2022,13(6):1641
Natural killer cells (NK cells) are important immune cells which have attracted increasing attention in cancer immunotherapy. Due to the heterogeneity of cells, individual cancer cells show different resistance to NK cytotoxicity, which has been revealed by flow cytometry. Here we used label-free mass cytometry (CyESI-MS) as a new tool to analyze the metabolites in Human Hepatocellular Carcinoma (HepG2) cells at the single-cell level after the interaction with different numbers of NK92 MI cells. A large amount of chemical information from individual HepG2 cells was obtained showing the process of cell apoptosis induced by NK cells. Nineteen metabolites which consecutively change during cell apoptosis were revealed by calculating their average relative intensity. Four metabolic pathways were impacted during cell apoptosis which hit 4 metabolites including glutathione (GSH), creatine, glutamic acid and taurine. We found that the HepG2 cells could be divided into two phenotypes after co-culturing with NK cells according to the bimodal distribution of concentration of these 4 metabolites. The correlation between metabolites and different apoptotic pathways in the early apoptosis cell group was established by the 4 metabolites at the single-cell level. This is a new idea of using single-cell specific metabolites to reveal the metabolic heterogeneity in cell apoptosis which would be a powerful means for evaluating the cytotoxicity of NK cells.Label-free mass cytometry is utilized to study the dynamic metabolic change during apoptosis in HepG2 cells induced by NK92 MI cells at the single-cell level. The metabolic heterogeneity of individual HepG2 cells during apoptosis was revealed. 相似文献
3.
Analysis of intracellular materials at the single-cell level presents opportunities for probing the heterogeneity of a cell population. Lysis by electroporation has been gaining popularity as a rapid method for disruption of the cell membrane and release of intracellular contents. In this report, we selectively released specific intracellular molecules for interrogation at the single-cell level by tuning the parameters of electroporation. We examined the release of a small molecule, calcein (MW approximately 600), and a 72-kDa protein kinase, Syk, tagged by enhanced green fluorescent protein (EGFP) from chicken B cells during electroporation at the single-cell level. We studied the effects of the field intensity and the field duration on the release of the two molecules. We found that calcein in general was released at lower field intensities and shorter durations than did SykEGFP. By tuning the electrical parameters, we were able to deplete calcein from the cells before SykEGFP started to release. This approach potentially provides a high-throughput alternative for probing different intracellular molecules at the single-cell level compared to chemical cytometry by eliminating complete disruption of the cell membrane. 相似文献
4.
The Tetrahymena trans-splicing ribozyme can edit RNA in a sequence-specific manner, but its efficiency needs to be improved for any functional rescues. This communication describes a simple method that uses a bacterial enzyme beta-lactamase to report trans-splicing activity of Tetrahymena ribozyme in single living mammalian cells by fluorescence microscopy and flow cytometry. This enzyme-based single-cell detection method is highly sensitive and compatible with living cell flow cytometry, and should allow a cell-based systematic screening of a vast library of ribozymes for better trans-spliced ribozyme variants. 相似文献
5.
Very little is known about the molecular mechanisms supporting living cell membrane electropermeabilization. This concept is based on the local membrane permeability induced by cell exposure to brief and intense external electric field pulses. During the electric field application, an electro-induced membrane electric potential difference is created that is locally associated with the dielectric properties of the plasma membrane. When the new membrane electric potential difference locally reaches a critical value, a local alteration of the membrane structure is induced and leads to reversible permeabilization. In our study, we attempted to determine whether mechanical tension could modulate the triggering of membrane electropermeabilization. Change in lateral tension of Chinese Hamster Ovary cell membrane has been osmotically induced. Cell electropermeabilization was performed in the minute time range after the osmotic stress, i.e., before the regulatory volume decrease being activated by the cell. Living cell electropermeabilization was analyzed on cell population using flow cytometry. We observed that electropermeabilization triggering was significantly facilitated when the lateral membrane tension was increased. The main conclusion is that the critical value of transmembrane potential needed to trigger membrane electropermeabilization, is smaller when the membrane is under lateral mechanical constraint. This supports the hypothesis that both mechanical and electrical constraints play a key role in transient membrane destabilization. 相似文献
6.
Flow cytometry (FCM) is a powerful technique for single-bacteria analysis via simultaneous light-scattering and fluorescence measurements. By offering high-throughput, quantitative, and multiparameter analysis at the single-cell level, FCM has gained an increased popularity in microbiological research, food safety monitoring, water quality control, and clinical diagnosis. Here we will review the recent applications of flow cytometry in areas such as (1) total bacterial cell count, (2) bacterial viability analysis, (3) specific bacterial detection and identification, (4) characterization of physiological changes under environmental perturbations, and (5) biological function studies. Nevertheless, despite these widespread applications, challenges still remain for the detection of small sizes of bacteria and biochemical features that cannot be brightly stained via fluorescence. Recent improvement in FCM instrumentation will be discussed, and particularly the development of high sensitivity flow cytometry for advanced analysis of single bacterial cells will be highlighted. 相似文献
7.
Xing Wei Meng Yang Ze Jiang Jinhui Liu Xuan Zhang Mingli Chen Jianhua Wang 《中国化学快报》2022,33(3):1373-1376
Accurate single-cell capture is a crucial step for single cell biological and chemical analysis. Conventional single-cell capturing often confront operational complexity, limited efficiency, cell damage, large scale but low accuracy, incompetence in the acquirement of nano-upgraded single-cell liquid. Flow cytometry has been widely used in large-scale single-cell detection, while precise single-cell isolation relies on both a precision operating platform and a microscope, which is not only extre... 相似文献
8.
9.
Christian Kappe Zainelabdeen H. Mohamed Eyad Naser Dr. Alexander Carpinteiro Prof. Dr. Christoph Arenz 《Chemistry (Weinheim an der Bergstrasse, Germany)》2020,26(26):5780-5783
Activity of acid sphingomyelinase has been implicated in a number of diseases like acute lung injury, sepsis or metastasis of melanoma cells. Here, we present a sphingomyelinase FRET probe based on FAM/BODIPY dyes for real-time monitoring of acid sphingomyelinase. The probe gives rise to a tremendous increase in fluorescence of the fluorescein FRET donor upon cleavage and we show that this is, to a significant part, due to cleavage-associated phase transition, suggesting a more systematic consideration of such effects for future probe development. The probe allows for the first time to monitor relative sphingomyelinase activities of intact living cells by flow cytometry. 相似文献
10.
We report a reliable strategy to perform automated image cytometry of single (non-adherent) stem cells captured in microfluidic traps. The method rapidly segments images of an entire microfluidic chip based on the detection of horizontal edges of microfluidic channels, from where the position of the trapped cells can be derived and the trapped cells identified with very high precision (>97%). We used this method to successfully quantify the efficiency and spatial distribution of single-cell loading of a microfluidic chip comprised of 2048 single-cell traps. Furthermore, cytometric analysis of trapped primary hematopoietic stem cells (HSC) faithfully recapitulated the distribution of cells in the G1 and S/G2-M phase of the cell cycle that was measured by flow cytometry. This approach should be applicable to automatically track single live cells in a wealth of microfluidic systems. 相似文献
11.
Jae Hyung Lee Seung Hwan Lee Sung Sun Yim Kyoung-Hee Kang Sang Yup Lee Si Jae Park Ki Jun Jeong 《Applied biochemistry and biotechnology》2013,170(7):1767-1779
Here, we report on a highly sensitive method for the detection of P(3HB) accumulation in Escherichia coli cells based on the automated flow cytometry system using fluorescent dyes. E. coli containing P(3HB) were stained with either BODIPY or Nile red fluorescent dye, and their staining properties were analyzed under a variety of conditions. Compared with Nile red, BODIPY was much more sensitive in staining P(3HB) and overall demonstrated a more rapid staining of cells, a greater resistance to photobleaching, and greater cell viability. In addition, we also successfully monitored heterogeneity in P(3HB) accumulation within a cell population using BODIPY staining and flow cytometry. We believe this optimized staining method using BODIPY in combination with screening by high-speed flow cytometer will be helpful in the engineering of host cells toward an enhanced production of bioplastics. 相似文献
12.
《Analytical letters》2012,45(6):944-957
Imatinib mesylate is a standard first-line therapy for patients with chronic myelogenous leukemia. However, there is still a significant proportion of these patients who reflect sub-optimal responses or fail imatinib therapy. Knowledge of the distribution within the studied system (e.g., peripheral blood) may be of high importance for understanding the principles of drug action and possible patient resistance to treatment. Intracellular or more precisely cell-associated, imatinib concentrations in patients, were shown to be higher compared to those in plasma, but still only limited data related to the methodology aspects of cell-associated concentrations are available. Herein is presented an assessment of the cell-associated imatinib determination assay by mass spectrometry. Three approaches were evaluated to isolate cells from the peripheral blood of chronic myelogenous leukemia patients. Erythrocyte lysis was found to cause substantial leakage of cell-associated imatinib in the first step. Selected alternative procedures utilizing density gradients did not affect the cell-associated imatinib concentration significantly. Cell isolates were subjected to flow cytometry which revealed differences in the population composition of peripheral blood cell isolates among individual patients indicating that the cell isolate composition should be addressed with the cell-associated imatinib concentration. The proposed approach may be utilized for the determination of intracellular concentration of imatinib and for other drugs in which the intracellular concentration plays a key role in the therapy. 相似文献
13.
A study to explore the optimal experimental parameters and the photosensitization of 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) in promyelocytic leukemia cell HL60 has been conducted, in which HL60 cells and their control groups, peripheral blood mononuclear cell (PBMC), first are incubated with different concentrations of ALA in dark for different periods of time and then followed by irradiating with different wavebands for different fluences. Fluorescence microscope and spectrofluorometer have been used to detect the fluorescence of protoporphyrin IX (PpIX) endogenously produced by ALA. The response of the cells to ALA-PDT was evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2-5-diphenyl-2H-tetrazolium bromide (MTT) assay (interval between irradiation and the MTT assay is 24 h) and by flow cytometry (the length of time between irradiation and the flow assay is 30 min). MTT results will reflect the relative number of metabolically active mitochondria in the population. Propidium iodide uptake in flow cytometry will test for membrane damage. The results of parameter experiments were obtained: 1 x 10(5)/mL HL60 cell was first incubated with 1 mmol/L ALA in dark for 4 h and the maximum fluorescence of PpIX level appeared; then irradiated with 410 nm (4 mW/cm2) for 14.4 J/cm2 and maximum photodamage to membrane and mitochondrial function of HL60 cell resulted. With the normal granulocytes, such response was not detected. Therefore a hypothetical idea can be brought forward that ALA-based PDT can be used for inactivation of leukemia cell HL60 and these optimal parameters may be useful for clinical application. 相似文献
14.
Azita Fazelkhah Samaneh Afshar Nicholas Durham Michael Butler Elham Salimi Greg Bridges Douglas Thomson 《Electrophoresis》2020,41(9):720-728
In this work, we present an optical transit DEP flow cytometer for parallel single-cell analysis. Each cell's dielectric property is inferred from velocity perturbations due to DEP actuation in a microfluidic channel. Dual LED sources facilitate velocity measurement by producing two transit shadows for each cell passing through the channel. These shadows are detected using a 256-pixel linear optical array detector. Massively parallel analysis is possible as each pixel of the detector can independently analyze the passing cells. A wide channel (∼18 mm) was employed to carry many particles simultaneously, and the system was capable of detecting the velocity of over 200 cells simultaneously. We have achieved analysis rates for 10 µm diameter polystyrene spheres response exceeding 250 per second. With appropriate calibration, this DEP cytometer can quantitatively measure the dielectric response. The dielectric response (Clausius–Mossotti factor) of viable CHO cells was measured over the frequency range of 100 kHz to 6 MHz, and the obtained response matches the previously measured values by our group. The DEP cytometer uses simple modular components to achieve high throughput label-free single-cell dielectric analysis and can begin analyzing particles within 10 s after starting to pump the sample into the channel. 相似文献
15.
Murat Serhatlioglu Mohammad Asghari Mustafa Tahsin Guler Caglar Elbuken 《Electrophoresis》2019,40(6):906-913
Elastic nature of the viscoelastic fluids induces lateral migration of particles into a single streamline and can be used by microfluidic based flow cytometry devices. In this study, we investigated focusing efficiency of polyethylene oxide based viscoelastic solutions at varying ionic concentration to demonstrate their use in impedimetric particle characterization systems. Rheological properties of the viscoelastic fluid and particle focusing performance are not affected by ionic concentration. We investigated the viscoelastic focusing dynamics using polystyrene (PS) beads and human red blood cells (RBCs) suspended in the viscoelastic fluid. Elasto‐inertial focusing of PS beads was achieved with the combination of inertial and viscoelastic effects. RBCs were aligned along the channel centerline in parachute shape which yielded consistent impedimetric signals. We compared our impedance‐based microfluidic flow cytometry results for RBCs and PS beads by analyzing particle transit time and peak amplitude at varying viscoelastic focusing conditions obtained at different flow rates. We showed that single orientation, single train focusing of nonspherical RBCs can be achieved with polyethylene oxide based viscoelastic solution that has been shown to be a good candidate as a carrier fluid for impedance cytometry. 相似文献
16.
M Wu TD Perroud N Srivastava CS Branda KL Sale BD Carson KD Patel SS Branda AK Singh 《Lab on a chip》2012,12(16):2823-2831
We have developed a microfluidic platform that enables, in one experiment, monitoring of signaling events spanning multiple time-scales and cellular locations through seamless integration of cell culture, stimulation and preparation with downstream analysis. A combination of two single-cell resolution techniques-on-chip multi-color flow cytometry and fluorescence imaging provides multiplexed and orthogonal data on cellular events. Automated, microfluidic operation allows quantitatively- and temporally-precise dosing leading to fine time-resolution and improved reproducibility of measurements. The platform was used to profile the toll-like receptor (TLR4) pathway in macrophages challenged with lipopolysaccharide (LPS)-beginning with TLR4 receptor activation by LPS, through intracellular MAPK signaling, RelA/p65 translocation in real time, to TNF-α cytokine production, all in one small macrophage population (< 5000 cells) while using minute reagent volume (540 nL/condition). The platform is easily adaptable to many cell types including primary cells and provides a generic platform for profiling signaling pathways. 相似文献
17.
A goal of modern biology is to understand the molecular mechanisms underlying cellular function. The ability to manipulate and analyze single cells is crucial for this task. The advent of microengineering is providing biologists with unprecedented opportunities for cell handling and investigation on a cell-by-cell basis. For this reason, lab-on-a-chip (LOC) technologies are emerging as the next revolution in tools for biological discovery. In the current discussion, we seek to summarize the state of the art for conventional technologies in use by biologists for the analysis of single, mammalian cells, and then compare LOC devices engineered for these same single-cell studies. While a review of the technical progress is included, a major goal is to present the view point of the practicing biologist and the advances that might increase adoption by these individuals. The LOC field is expanding rapidly, and we have focused on areas of broad interest to the biology community where the technology is sufficiently far advanced to contemplate near-term application in biological experimentation. Focus areas to be covered include flow cytometry, electrophoretic analysis of cell contents, fluorescent-indicator-based analyses, cells as small volume reactors, control of the cellular microenvironment, and single-cell PCR. 相似文献
18.
van den Brink FT Gool E Frimat JP Bomer J van den Berg A Le Gac S 《Electrophoresis》2011,32(22):3094-3100
We report a PDMS microfluidic platform for parallel single-cell analysis (PaSCAl) as a powerful tool to decipher the heterogeneity found in cell populations. Cells are trapped individually in dedicated pockets, and thereafter, a number of invasive or non-invasive analysis schemes are performed. First, we report single-cell trapping in a fast (2-5 min) and reproducible manner with a single-cell capture yield of 85% using two cell lines (P3x63Ag8 and MCF-7), employing a protocol which is scalable and easily amenable to automation. Following this, a mixed population of P3x63Ag8 and MCF-7 cells is stained in situ using the nucleic acid probe (Hoechst) and a phycoerythrin-labeled monoclonal antibody directed at EpCAM present on the surface of the breast cancer cells MCF-7 and absent on the myeloma cells P3x63Ag8 to illustrate the potential of the device to analyze cell population heterogeneity. Next, cells are porated in situ using chemicals in a reversible (digitonin) or irreversible way (lithium dodecyl sulfate). This is visualized by the transportation of fluorescent dyes through the membrane (propidium iodide and calcein). Finally, an electrical protocol is developed for combined cell permeabilization and electroosmotic flow (EOF)-based extraction of the cell content. It is validated here using calcein-loaded cells and visualized through the progressive recovery of calcein in the side channels, indicating successful retrieval of individual cell content. 相似文献
19.
Optical microscopy of single bacteria growing on solid agarose support is a powerful method for studying the natural heterogeneity in growth and gene expression. While the material properties of agarose make it an excellent substrate for such studies, the sheer number of exponentially growing cells eventually overwhelms the agarose pad, which fundamentally limits the duration and the throughput of measurements. Here we overcome the limitations of exponential growth by patterning agarose pads on the sub-micron-scale. Linear tracks constrain the growth of bacteria into a high density array of linear micro-colonies. Buffer flow through microfluidic lines washes away excess cells and delivers fresh nutrient buffer. Densely patterned tracks allow us to cultivate and image hundreds of thousands of cells on a single agarose pad over 30-40 generations, which drastically increases single-cell measurement throughput. In addition, we show that patterned agarose can facilitate single-cell measurements within bacterial communities. As a proof-of-principle, we study a community of E. coli auxotrophs that can complement the amino acid deficiencies of one another. We find that the growth rate of colonies of one strain decreases sharply with the distance to colonies of the complementary strain over distances of only a few cell lengths. Because patterned agarose pads maintain cells in a chemostatic environment in which every cell can be imaged, we term our device the single-cell chemostat. High-throughput measurements of single cells growing chemostatically should greatly facilitate the study of a variety of microbial behaviours. 相似文献
20.
The gene expression of human embryonic stem cells (hESC) is a critical aspect for understanding the normal and pathological development of human cells and tissues. Current bulk gene expression assays rely on RNA extracted from cell and tissue samples with various degree of cellular heterogeneity. These 'cell population averaging' data are difficult to interpret, especially for the purpose of understanding the regulatory relationship of genes in the earliest phases of development and differentiation of individual cells. Here, we report a microfluidic approach that can extract total mRNA from individual single-cells and synthesize cDNA on the same device with high mRNA-to-cDNA efficiency. This feature makes large-scale single-cell gene expression profiling possible. Using this microfluidic device, we measured the absolute numbers of mRNA molecules of three genes (B2M, Nodal and Fzd4) in a single hESC. Our results indicate that gene expression data measured from cDNA of a cell population is not a good representation of the expression levels in individual single cells. Within the G0/G1 phase pluripotent hESC population, some individual cells did not express all of the 3 interrogated genes in detectable levels. Consequently, the relative expression levels, which are broadly used in gene expression studies, are very different between measurements from population cDNA and single-cell cDNA. The results underscore the importance of discrete single-cell analysis, and the advantages of a microfluidic approach in stem cell gene expression studies. 相似文献