A lipidomics demonstration of the importance of single cell analysis

Phelps, Mandy S.; Verbeck, Guido F.

doi:10.1039/c5ay00379b

Cited by 26 publications

(26 citation statements)

References 23 publications

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“…In human breast tissue, cancer associated adipocytes (CAAs) and adjacent healthy adipocytes revealed distinct differences in heterogeneity of TAG molecular species composition from extractions using a two-tip method with the nanomanipulator using NSI [14]. Vastly different lipid profiles were also obtained dependent on lipid droplet size from whole cell digestions of differentiated human adipocytes using NSI, which validated the workstation as a valuable tool for in vitro cell cultures [18]. Using microextractions and NSI for biological work has proven to be advantageous in several experiments, but is not without issues and areas for improvement.…”

Section: Introductionmentioning

confidence: 92%

Nanomanipulation-Coupled Matrix-Assisted Laser Desorption/ Ionization-Direct Organelle Mass Spectrometry: A Technique for the Detailed Analysis of Single Organelles

Phelps

Chapman

Verbeck

2015

J. Am. Soc. Mass Spectrom.

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Abstract. We describe a novel technique combining precise organelle microextraction with deposition and matrix-assisted laser desorption/ionization (MALDI) for a rapid, minimally invasive mass spectrometry (MS) analysis of single organelles from living cells. A dual-positioner nanomanipulator workstation was utilized for both extraction of organelle content and precise co-deposition of analyte and matrix solution for MALDI-direct organelle mass spectrometry (DOMS) analysis. Here, the triacylglycerol (TAG) profiles of single lipid droplets from 3T3-L1 adipocytes were acquired and results validated with nanoelectrospray ionization (NSI) MS. The results demonstrate the utility of the MALDI-DOMS technique as it enabled longer mass analysis time, higher ionization efficiency, MS imaging of the co-deposited spot, and subsequent MS/MS capabilities of localized lipid content in comparison to NSI-DOMS. This method provides selective organellar resolution, which complements current biochemical analyses and prompts for subsequent subcellular studies to be performed where limited samples and analyte volume are of concern.

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Section: Introductionmentioning

confidence: 92%

Nanomanipulation-Coupled Matrix-Assisted Laser Desorption/ Ionization-Direct Organelle Mass Spectrometry: A Technique for the Detailed Analysis of Single Organelles

Phelps

Chapman

Verbeck

2015

J. Am. Soc. Mass Spectrom.

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“…Thus, single-cell analysis of lipids in thrombocytes with a nanomanipulator coupled to mass spectrometry appears to be highly sensitive. [12][13][14] In our analysis, because we used 2 stages of thrombocyte development, we used PC as a control, and lipids were represented as a relative percentage of this control PC. Interestingly, several classes of lipids ( Figure 4C) showed identical percentages.…”

Section: Discussionmentioning

confidence: 99%

“…The contents were vortexed, and the diluted blood sample was pipetted to fill the depression slide. The 3 thrombocytes were extracted using an L200 nanomanipulator workstation (DCG Systems, Fremont, CA), [12][13][14] which was mounted to an inverted Nikon TE2000-U microscope and is equipped with a C-HGFI fluorescence source for fluorescence microscopy. The nanomanipulator is connected to a 60-psi nitrogen gas pressure injector (MicroData Instrument, South Plainfield, NJ) with 2 nanopositioners.…”

Section: Cell Extraction and Maldi-ltq-orbitrap Analysismentioning

confidence: 99%

“…The nanomanipulator is connected to a 60-psi nitrogen gas pressure injector (MicroData Instrument, South Plainfield, NJ) with 2 nanopositioners. 14 The capillary tip in the nanopositioner was backfilled with 8 mL of a 2:1 chloroform/methanol (vol/vol) solution. The matrix solution for matrix-assisted laser desorption/ionization (MALDI), 8 mL of 20 mg/mL 2,5 dihydroxybenzoic acid solution in 3:2 acetonitrile/water (vol/vol), was backloaded to the second nanopositioner.…”

Section: Cell Extraction and Maldi-ltq-orbitrap Analysismentioning

confidence: 99%

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Generation of transgenic zebrafish with 2 populations of RFP- and GFP-labeled thrombocytes: analysis of their lipids

Fallatah¹,

Silva

Verbeck

et al. 2019

Blood Advances

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Zebrafish thrombocytes are similar to mammalian platelets. Mammals have young platelets (also called reticulated platelets) and mature platelets. Likewise, zebrafish have 2 populations of thrombocytes; one is DiI-C18 (DiI)+ (DP), and the other is DiI− (DN). However, the mechanism of selective thrombocyte labeling by DiI is unknown. Furthermore, there is no transgenic zebrafish line where DP and DN thrombocytes are differentially labeled with fluorescent proteins. In this study, we found that Glo fish, in which the myosin light chain 2 promoter drives the rfp gene, have a population of thrombocytes that are red fluorescent protein (RFP) labeled. We also generated transgenic GloFli fish in which DP and DN thrombocytes are labeled with RFP and green fluorescent protein (GFP), respectively. Single-cell lipid analysis showed a twofold increase in phosphatidylethanolamine (PE) and a twofold decrease in phosphatidylcholine (PC) in RFP+ thrombocytes compared with GFP+ thrombocytes, suggesting that lipid composition may be important for DiI differential labeling. Therefore, we tested liposomes prepared with different ratios of PC and PE and observed that liposomes prepared with higher amounts of PE favor DiI labeling, whereas the PC concentration had a modest effect. In liposomes prepared using only PE or PC, increased concentrations of PE resulted in increased DiI binding. These results suggest that because RFP+ thrombocytes have higher PE concentrations, DiI may bind to them efficiently, thus explaining the selective labeling of thrombocytes by DiI. This work also provides GloFli fish that should be useful in understanding the mechanism of thrombocyte maturation.

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“…It is important to understand the interactions between different organs as well as interactions between cells especially for disease studies and drug development and checking drug efficacy. There has also been an increasing realization of the heterogeneity between cells of the same cell type 88 cell analysis will also be helpful in understanding the make-up of rare cells like the cancer stem cells about which not much understanding is currently available.…”

Section: I) Single Cell Analysismentioning

confidence: 99%

Microfluidic in vitro model to study cell mechanisms and drug efficacy

Menon¹

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In vitro models are commonly used biological tools involving culture of cells so as to replicate complex human organs and organ systems in an ex-vivo manner. Researchers have created in vitro models for many years now focusing on different physiologically relevant studies. Non-invasiveness and simplicity are some of the major advantages of in vitro models unlike in the case of animal models where animals are sacrificed and moreover the complexity of the animal physiology introduces a large number of variables thereby complicating the entire study. An in vitro model reduces the number of variables and hence simplifies the study. One of the techniques for the creation of in vitro models is cell patterning. It involves the spatial orientation of the cells in a Abstract XII where the co-culture model developed using this device was used to test the targeted killing potential of a photosensitizer that was designed to perform targeted photodynamic therapy of cancer cells. Apart from performing cell culture, the microfluidic chip also allows for easier analysis using suitable fluorescent probes. In this thesis we have discussed about a simple, easy to use and highly reproducible microfluidic model that was developed to perform cell culture and cellular analysis on chip according to the study of interest. Apart from cell mechanism studies, the model can be modified to perform other studies like testing drug selectivity, drug efficacy as well as for performing preliminary tests during hypothesis testing.

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A lipidomics demonstration of the importance of single cell analysis

Abstract: A novel single cell analytical method showing adipocyte heterogeneity dependent on lipid droplet size using nanomanipulation-coupled nanoelectrospray mass spectrometry.

Cited by 26 publications

References 23 publications

Nanomanipulation-Coupled Matrix-Assisted Laser Desorption/ Ionization-Direct Organelle Mass Spectrometry: A Technique for the Detailed Analysis of Single Organelles

Nanomanipulation-Coupled Matrix-Assisted Laser Desorption/ Ionization-Direct Organelle Mass Spectrometry: A Technique for the Detailed Analysis of Single Organelles

Generation of transgenic zebrafish with 2 populations of RFP- and GFP-labeled thrombocytes: analysis of their lipids

Microfluidic in vitro model to study cell mechanisms and drug efficacy

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