Single cell lipidome analysis of phosphatidylcholines and spingomyelins from prostate cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Human Homo Sapiens
TISSUE(S): Cultured Cells
SUBMITTER: 
Sarah Hancock
PROVIDER: ST002367 | MetabolomicsWorkbench | Sun Oct 30 00:00:00 BST 2022
REPOSITORIES: MetabolomicsWorkbench
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| mwtab | Other |
Items per page: 5 1 - 1 of 1 |
Similar Datasets
Project description:We have developed a high-throughput method for the detection and quantification of a wide range of phosphatidylcholine (PC) and sphingomyelin (SM) species from single cells that combines fluorescence-assisted cell sorting (FACS) with automated chip-based nanoelectrospray ionization (nanoESI) and shotgun lipidomics. Using this method we can detect and perform relative quantitation on more than >50 different PC and SM species from immortalised human cells, and can easily distinguish between cells of different lineages (e.g. hepatocarcinoma HePG2 vs C2C12 myoblasts) and cells treated with exogenous fatty acids.
2022-10-19 | ST002366 | MetabolomicsWorkbench
Project description:We have enabled ultrasensitive proteomics of limited protein digests by developing a tapered-tip nanoelectrospray ionization (nanoESI) interface for microanalytical capillary electrophoresis (CE) high resolution mass spectrometry (HRMS). CE-nanoESI-HRMS allowed us to detect 260 zmol angiotensin II (lower limit of detection) and identify 1 pg of bovine serum albumin and cytochrome c. A 1 ng protein digest from the mouse posterior cortex yielded 217 nonredundant protein groups, including many gene products that are used as classical neuronal markers.
2022-02-18 | PXD005222 | Pride
Project description:Fluorescence-activated cell sorting (FACS) is a sensitive and valuable technique to characterize cellular subpopulations and great advances have been made using this approach. Cells are often fixed with formaldehyde prior to the sorting process to preserve cell morphology and maintain the expression of surface molecules, as well as to ensure safety in the sorting of infected cells. It is widely recognized that formaldehyde fixation alters RNA and DNA structure and integrity, thus analyzing gene expression in these cells has been difficult. We therefore examined the effects of formaldehyde fixation on the stability and quantitation of nucleic acids in cell lines, primary leukocytes and also cells isolated from SIV-infected pigtailed macaques. We developed a method to extract RNA from fixed cells that yielded the same amount of RNA as our common method of RNA isolation from fresh cells. Quantitation of RNA by RT-qPCR in fixed cells was not always comparable with that in unfixed cells. In comparison, when RNA was measured by the probe-based NanoString system, there was no significant difference in RNA quantitation. In addition, we demonstrated that quantitation of proviral DNA in fixed cells by qPCR is comparable to that in unfixed cells when normalized by a single-copy cellular gene. These results provide a systematic procedure to quantitate gene expression in cells that have been fixed with formaldehyde and sorted by FACS.
2013-09-09 | GSE50690 | GEO
Project description:Fluorescence-activated cell sorting (FACS) is a sensitive and valuable technique to characterize cellular subpopulations and great advances have been made using this approach. Cells are often fixed with formaldehyde prior to the sorting process to preserve cell morphology and maintain the expression of surface molecules, as well as to ensure safety in the sorting of infected cells. It is widely recognized that formaldehyde fixation alters RNA and DNA structure and integrity, thus analyzing gene expression in these cells has been difficult. We therefore examined the effects of formaldehyde fixation on the stability and quantitation of nucleic acids in cell lines, primary leukocytes and also cells isolated from SIV-infected pigtailed macaques. We developed a method to extract RNA from fixed cells that yielded the same amount of RNA as our common method of RNA isolation from fresh cells. Quantitation of RNA by RT-qPCR in fixed cells was not always comparable with that in unfixed cells. In comparison, when RNA was measured by the probe-based NanoString system, there was no significant difference in RNA quantitation. In addition, we demonstrated that quantitation of proviral DNA in fixed cells by qPCR is comparable to that in unfixed cells when normalized by a single-copy cellular gene. These results provide a systematic procedure to quantitate gene expression in cells that have been fixed with formaldehyde and sorted by FACS. We compared RNA quantitation between unfixed and formaldehyde-fixed cells using the NanoString nCounter system. This analysis was performed using two cell lines, K562 and CEMx174, and human PBMCs. Three biological replicates were performed for each cell type. We also performed this comparison using human PBMCs sorted by FACS. For this analysis, we isolated PBMCs from two donors and performed two technical replicates for each donor sample.
2013-09-09 | E-GEOD-50690 | biostudies-arrayexpress
Project description:Background: The microbiome is increasingly being linked to cancer risk. Little is known about the lung and oral cavity microbiomes in healthy smokers (SM), and even less for electronic cigarette (EC) users, compared healthy never-smokers (NS). Methods: In a cross-sectional pilot study of SM (N=8), EC users (N=10) and NS (N=10) saliva and bronchoscopy-collected bronchoalveolar lavage samples were collected. Bacteria species were identified through metatranscriptome profiling by RNA-sequencing to study associations with the lung and oral microbiome. Pairwise comparisons and linear modeling was assessed with false discovery rates <0.1. Results: Total bacterial load was similar for the SM, EC users and NS, and there was no differences in the bacterial diversity across groups. In the lung, there were 44 bacterial species that were statistically significantly different for SM/NS, 80% of which were decreased in the SM. There were 12 bacterial species that were different for SM/EC users, all of which were decreased, 10 of which were also identified in the SM/NS comparison. The 2 bacterial species unique to SM/EC comparison were Neisseria sp. KEM232 and Curvibacter sp. AEP1-3. From the top 5 decreased species in SM/EC, 3 were also identified in the SM/NS comparison (Neisseria elongata, Neisseria sicca, and Haemophilus parainfluenzae) and 2 of these were unique to the SM/EC comparison (Neisseria zoodegmatis and Ottowia sp. oral taxon 894). There were 8 species increased in SM compared to NS, none of which are known to be clinically significant. In the oral microbiome, 152 bacteria species were differentially abundant for the SM/NS analysis, and only 17 for the EC/NS comparison, all which were also present in SM/NS comparisons. There were 21 bacteria that were differentially abundant in both the lung and oral cavity for SM and NS, 95% also were decreased in the SM. Conclusion: Smoking and EC use do not appear to materially affect the lung microbiome, although differences are noted of unclear clinical significance. Most differentially abundant bacteria decreased, which may be due to a toxic effect of cigarette smoke, including a change in humidity or heating. Given the low number of overlapping oral and lung microbes, the oral microbiome does not appear to be a good surrogate for smoking-related effects in the lung.
2022-07-13 | GSE180785 | GEO
Project description:Fluorescence-activated cell sorting (FACS) is a specialized technique to isolate
cell subpopulations with a high level of recovery and accuracy. However, the cell sorting
procedure can impact the viability and metabolic state of cells. Here, we performed a comparative study and evaluated the impact of traditional high-pressure charged droplet-based and a microfluidic chip-based sorting approach on the metabolic and phosphoproteomic profile of different cell types. While microfluidic chip-based sorted cells more closely resembled the unsorted control group for most cell types tested, the droplet-based sorted cells showed significant metabolic and phosphoproteomic alterations. In particular, greater changes in redox and energy status were present in cells sorted with the droplet-based cell sorter along with higher transcriptional and spliceosomal regulation and mechanical stress signaling. These results
indicate microfluidic chip-based sorting is less disruptive compared to droplet-based sorting.
2023-11-09 | MSV000093348 | MassIVE
Project description:BLaER1 cells are human leukemia pre-B cells able to transdifferentiate into functional and non-tumorigenic macrophages. With the goal of uncovering the genes involved in the transdifferentiation process, we have designed a DECKO (Double Excision CRISPR Knockout) library to knockout lncRNAs and protein coding genes (pc-genes) overexpressed along the seven days the process lasts. We have seen that targeting pc-genes with two gRNAs synergistically enhances the efficiency of knock out, by inducing deletions and/or frameshifts that promote the expression of non-functional proteins. Thus, using the CRISPETa tool, we designed paired guide RNAs targeting either the region surrounding the Transcription Start Site of the 166 lncRNAs or the coding exons of the 874 pc candidate genes. Cas9-expressing BLaER1 cells were infected at low-multiplicity of infection with the combined library and induced transdifferentiation. Delayed and differentiated subpopulations of cells were isolated by Fluorescence-Activated Cell Sorting at 3 days and 6 days after induction, and pgRNAs were sequenced in an Illumina HiSeq2500 instrument. The sequencing reads were mapped and quantified to uncover the enriched pgRNAs found in each subpopulation. Among all genes targeted in the library, we identified twenty pc-genes and six lncRNAs as strong candidates to be involved in the process, as the pgRNAs targeting their loci were enriched in the delayed population compared to the differentiated one, either at 3 days or at 6 days after transdifferentiation induction. From these, we selected two pc-genes and two lncRNAs for deeper characterization.
2022-04-25 | E-MTAB-10445 | biostudies-arrayexpress
Project description:Prostate cancer (PCa) is the most common cancer in American men. The American Cancer Society’s estimates for prostate cancer in the United States for 2017 are estimated 161.360 new cases and 26,730 deaths from PCa. To study metastatic properties to bone, PC-3 cell line is mainly used classical human prostatic carcinoma cell line, established and characterized its tumorigenicity from a human prostatic adenocarcinoma metastatic to bone is reported. In addition, PC-3/nkR cell line, natural killer(NK) cells-resistant, was isolated from mammary tumor xenograft studies in mice from PC-3 was implanted to nude mice and fecund to be tumorigenic in the early 2000s. In this study, we investigated secreted proteins of the conditioned media of PC-3 and PC-3/nkR cell lines using comparative proteomics technology to identify the molecular mechanism related to metastatic processes related to PC-3/nkR. Our study showed PC-3/nkR cells are new highly migrated and NK cells-resistant cell-line compared to PC-3 cells, as novel highly malignant tumor cells to study mechanisms of PCa metastatic.
2019-08-19 | PXD008184 | Pride
Project description:We developed a new method for ChIP-seq with per-cell normalization (pc-ChIP-seq) to define PAX3-FOXO1 localization in rhabdomyosarcoma (RMS). We report novel pioneer function for the major driver oncogene in RMS, with nucleosome-motif targeting and kinetic displacement of nucleosomes in human cells.
2020-12-12 | GSE163068 | GEO