scholarly journals CRISPRs for Optimal Targeting: Delivery of CRISPR Components as DNA, RNA, and Protein into Cultured Cells and Single-Cell Embryos

2016 ◽  
Vol 27 (6) ◽  
pp. 464-475 ◽  
Author(s):  
Evguenia Kouranova ◽  
Kevin Forbes ◽  
Guojun Zhao ◽  
Joe Warren ◽  
Angela Bartels ◽  
...  
Keyword(s):  
Single Cell ◽  
Cultured Cells ◽  
Targeting Delivery

scholarly journals Single cell RNA sequencing of calvarial and long bone endocortical cells

2019 ◽  
Author(s):  
Ugur M. Ayturk ◽  
Joseph P. Scollan ◽  
Alexander Vesprey ◽  
Christina M. Jacobsen ◽  
Paola Divieti Pajevic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cultured Cells ◽  
Neutralizing Antibody ◽  
Long Bone ◽  
Appendicular Skeleton ◽  
Rna Seq ◽  
Isolated Cells

ABSTRACTSingle cell RNA-seq (scRNA-seq) is emerging as a powerful technology to examine transcriptomes of individual cells. We determined whether scRNA-seq could be used to detect the effect of environmental and pharmacologic perturbations on osteoblasts. We began with a commonly used in vitro system in which freshly isolated neonatal mouse calvarial cells are expanded and induced to produce a mineralized matrix. We used scRNA-seq to compare the relative cell type abundances and the transcriptomes of freshly isolated cells to those that had been cultured for 12 days in vitro. We observed that the percentage of macrophage-like cells increased from 6% in freshly isolated calvarial cells to 34% in cultured cells. We also found that Bglap transcripts were abundant in freshly isolated osteoblasts but nearly undetectable in the cultured calvarial cells. Thus, scRNA-seq revealed significant differences between heterogeneity of cells in vivo and in vitro. We next performed scRNA-seq on freshly recovered long bone endocortical cells from mice that received either vehicle or Sclerostin-neutralizing antibody for 1 week. Bone anabolism-associated transcripts were also not significantly increased in immature and mature osteoblasts recovered from Sclerostin-neutralizing antibody treated mice; this is likely a consequence of being underpowered to detect modest changes in gene expression, since only 7% of the sequenced endocortical cells were osteoblasts, and a limited portion of their transcriptomes were sampled. We conclude that scRNA-seq can detect changes in cell abundance, identity, and gene expression in skeletally derived cells. In order to detect modest changes in osteoblast gene expression at the single cell level in the appendicular skeleton, larger numbers of osteoblasts from endocortical bone are required.

scholarly journals Single-cell transcriptional dynamics of flavivirus infection

2017 ◽  
Author(s):  
Fabio Zanini ◽  
Szu-Yuan Pu ◽  
Elena Bekerman ◽  
Shirit Einav ◽  
Stephen R. Quake
Keyword(s):  
Single Cell ◽  
Membrane Trafficking ◽  
Viral Infections ◽  
Cultured Cells ◽  
Complex Dynamics ◽  
Single Cell Level ◽  
Cell Level ◽  
Transcriptional Dynamics ◽  
High Degree ◽  
Virus Abundance

ABSTRACTDengue and Zika viral infections affect millions of people annually and can be complicated by hemorrhage or neurological manifestations, respectively. However, a thorough understanding of the host response to these viruses is lacking, partly because conventional approaches ignore heterogeneity in virus abundance across cells. We present viscRNA-Seq (virus-inclusive single cell RNA-Seq), an approach to probe the host transcriptome together with intracellular viral RNA at the single cell level. We applied viscRNA-Seq to monitor dengue and Zika virus infection in cultured cells and discovered extreme heterogeneity in virus abundance. We exploited this variation to identify host factors that show complex dynamics and a high degree of specificity for either virus, including proteins involved in the endoplasmic reticulum translocon, signal peptide processing, and membrane trafficking. We validated the viscRNA-Seq hits and discovered novel proviral and antiviral factors. viscRNA-Seq is a powerful approach to assess the genome-wide virus-host dynamics at single cell level.

scholarly journals Culture and Characterization of Microglia from the Adult Murine Retina

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Gayathri Devarajan ◽  
Mei Chen ◽  
Elizabeth Muckersie ◽  
Heping Xu
Keyword(s):  
Single Cell ◽  
Proinflammatory Cytokines ◽  
Cultured Cells ◽  
Calf Serum ◽  
Culture Conditions ◽  
Simple Method ◽  
Mhc Ii ◽  
Adult Mice ◽  
Retinal Microglia ◽  
And Function

Purpose. To develop a protocol for isolating and culturing murine adult retinal microglia and to characterize the phenotype and function of the cultured cells.Method. Retinal single-cell suspensions were prepared from adult MF1 mice. Culture conditions including culture medium, growth factors, seeding cell density, and purification of microglia from the mixed cultures were optimised. Cultured retinal microglial cells were phenotyped using the surface markers CD45, CD11b, and F4/80. Their ability to secrete proinflammatory cytokines in response to lipopolysaccharide (LPS) stimulation was examined using cytometric bead array (CBA) assay.Results. Higher yield was obtained when retinal single-cell suspension was cultured at the density of0.75×106cells per cm2in Dulbecco’s modified Eagle medium (DMEM)/F12 + Glutamax supplement with 20% fetal calf serum (FCS) and 20% L929 supernatant. We identified day 10 to be the optimum day of microglial isolation. Over 98% of the cells isolated were positive for CD45, CD11b, and F4/80. After stimulating with LPS they were able to secrete proinflammatory cytokines such as IL-6 and TNF-αand express CD86, CD40, and MHC-II.Conclusion. We have developed a simple method for isolating and culturing retinal microglia from adult mice.

scholarly journals High-throughput single-cell joint analysis of histone modifications and gene expression by Paired-Tag

2021 ◽  
Author(s):  
Chenxu Zhu ◽  
Yanxiao Zhang ◽  
Yang Eric Li ◽  
Jacinta Lucero ◽  
M. Margarita Behrens ◽  
...  
Keyword(s):  
Gene Expression ◽  
Molecular Biology ◽  
Single Cell ◽  
Histone Modifications ◽  
High Throughput ◽  
Cultured Cells ◽  
Single Cells ◽  
Joint Analysis ◽  
Gene Expressions ◽  
Standard Molecular Biology

Abstract We describe here Paired-Tag, a high-throughput multi-omics method for joint profiling of histone modifications and gene expressions in single cells. The assay is based on a combinatorial barcoding indexing strategy that does not require special instruments. It can be performed with nuclei extracted from cultured cells or frozen tissues, in standard molecular biology laboratories.

scholarly journals Single-cell analysis of upper airway cells reveals host-viral dynamics in influenza infected adults

2020 ◽  
Author(s):  
Yuming Cao ◽  
Zhiru Guo ◽  
Pranitha Vangala ◽  
Elisa Donnard ◽  
Ping Liu ◽  
...  
Keyword(s):  
Single Cell ◽  
Mhc Class Ii ◽  
Cultured Cells ◽  
Single Cell Analysis ◽  
Influenza Infection ◽  
Cell Types ◽  
Human Infection ◽  
Specific Cell ◽  
Natural Setting ◽  
Healthy Human

AbstractInfluenza virus infections are major causes of morbidity and mortality. Research using cultured cells, bulk tissue, and animal models cannot fully capture human disease dynamics. Many aspects of virus-host interactions in a natural setting remain unclear, including the specific cell types that are infected and how they and neighboring bystander cells contribute to the overall antiviral response. To address these questions, we performed single-cell RNA sequencing (scRNA-Seq) on cells from freshly collected nasal washes from healthy human donors and donors diagnosed with acute influenza during the 2017-18 season. We describe a previously uncharacterized goblet cell population, specific to infected individuals, with high expression of MHC class II genes. Furthermore, leveraging scRNA-Seq reads, we obtained deep viral genome coverage and developed a model to rigorously identify infected cells that detected influenza infection in all epithelial cell types and even some immune cells. Our data revealed that each donor was infected by a unique influenza variant and that each variant was separated by at least one unique non-synonymous difference. Our results demonstrate the power of massively-parallel scRNA-Seq to study viral variation, as well as host and viral transcriptional activity during human infection.

Restitution of single-cell defects in the mouse colon epithelium differs from that of cultured cells

2006 ◽  
Vol 290 (6) ◽  
pp. R1496-R1507 ◽  
Author(s):  
D. Günzel ◽  
P. Florian ◽  
J. F. Richter ◽  
H. Troeger ◽  
J. D. Schulzke ◽  
...  
Keyword(s):  
Single Cell ◽  
Laser Scanning ◽  
Pathogenic Bacteria ◽  
Cultured Cells ◽  
Healing Process ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Rapid Repair ◽  
E Coli ◽  
Tnf Α

Integrity of colon epithelium is of crucial importance and, as small defects occur constantly, rapid repair (restitution) is essential. To investigate the mechanism of restitution, single-cell lesions were induced in mouse colonic surface epithelia by iontophoretic injection of Ca2+. Closure of the resulting defects was monitored using confocal laser scanning microscopy (CLSM), and functional sealing by electrophysiological techniques. Restitution was evaluated as the time constant τ of the exponential decrease in conductance of an induced leak and amounted to 0.28 min under control conditions. After 4 min, the leak was completely sealed. Repair was thus considerably faster than in previously investigated HT-29/B6 cells (τ = 5.73 min). As in cultured cells, cytochalasin D delayed restitution in native colon epithelia (τ = 0.69 min), indicating the involvement of actin in the healing process; however, no accumulation of actin surrounding the lesion was detected. Long-term incubation of epithelia with IFN-γ alone or in combination with TNF-α increased τ to 0.49 and 0.59 min, respectively. In contrast to cultured cells, TNF-α alone did not affect restitution. A brief (<10 min) exposure to the sterile filtered supernatant of hemolytic E. coli O4 cultures did not affect the morphology of the epithelium, but delayed restitution. In CLSM studies, defects were still clearly visible 4 min after the onset of lesion induction. The supernatant of a nonhemolytic E. coli O4 mutant did not exhibit this effect. In conclusion, single-cell defects in native colon cause functional leaks that seal faster than in cell cultures. Proinflammatory cytokines and pathogenic bacteria delay restitution. This suggests a key role of very small lesions at the onset of pathogenic processes in the intestine.

scholarly journals Microfluidic Cell Trapping for Single-Cell Analysis

Micromachines ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 409 ◽  
Author(s):  
Bing Deng ◽  
Heyi Wang ◽  
Zhaoyi Tan ◽  
Yi Quan
Keyword(s):  
Fluid Dynamics ◽  
Cell Growth ◽  
Single Cell ◽  
Microfluidic Chip ◽  
Cultured Cells ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Cell Analysis ◽  
Cell Capture ◽  
Culture Process

The single-cell capture microfluidic chip has many advantages, including low cost, high throughput, easy manufacturing, integration, non-toxicity and good stability. Because of these characteristics, the cell capture microfluidic chip is increasingly becoming an important carrier on the study of life science and pharmaceutical analysis. Important promises of single-cell analysis are the paring, fusion, disruption and analysis of intracellular components for capturing a single cell. The capture, which is based on the fluid dynamics method in the field of micro fluidic chips is an important way to achieve and realize the operations mentioned above. The aim of this study was to compare the ability of three fluid dynamics-based microfluidic chip structures to capture cells. The effects of cell growth and distribution after being captured by different structural chips and the subsequent observation and analysis of single cells on the chip were compared. It can be seen from the experimental results that the microfluidic chip structure most suitable for single-cell capture is a U-shaped structure. It enables single-cell capture as well as long-term continuous culture and the single-cell observation of captured cells. Compared to the U-shaped structure, the cells captured by the microcavity structure easily overlapped during the culture process and affected the subsequent analysis of single cells. The flow shortcut structure can also be used to capture and observe single cells, however, the shearing force of the fluid caused by the chip structure is likely to cause deformation of the cultured cells. By comparing the cell capture efficiency of the three chips, the reagent loss during the culture process and the cell growth state of the captured cells, we are provided with a theoretical support for the design of a single-cell capture microfluidic chip and a reference for the study of single-cell capture in the future.

Microfabrication in silicon microphysiometry

1994 ◽  
Vol 40 (9) ◽  
pp. 1800-1804 ◽  
Author(s):  
G T Baxter ◽  
L J Bousse ◽  
T D Dawes ◽  
J M Libby ◽  
D N Modlin ◽  
...  
Keyword(s):  
Single Cell ◽  
Clinical Relevance ◽  
Cultured Cells ◽  
Effective Means ◽  
Antibiotic Sensitivity ◽  
Functional Assay ◽  
Physiological Changes ◽  
Cellular Receptors ◽  
The Past ◽  
Light Addressable Potentiometric Sensor

Abstract Over the past 5 years, microphysiometry has proved an effective means for detecting physiological changes in cultured cells, particularly as a functional assay for the activation of many cellular receptors. To demonstrate the clinical relevance of this method, we have used it to detect bacterial antibiotic sensitivity and to discriminate between bacteriostatic and bacteriocidal concentrations. The light-addressable potentiometric sensor, upon which microphysiometry is based, is well suited for structural manipulations based on photolithography and micromachining, and we have begun to take advantage of this capability. We present results from a research instrument with eight separate assay channels on a 5-cm2 chip. We discuss the planned evolution of the technology toward high-through-put instruments and instruments capable of performing single-cell measurements.

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