Glutaraldehyde fixation method for single‐cell lipid analysis by time‐of‐flight secondary ion‐mass spectrometry

Nagata, Yasuyuki; Ishizaki, Itsuko; Waki, Michihiko; Ide, Yoshimi; Hossen, Amir; Ohnishi, Kazunori; Sanada, N.; Setou, Mitsutoshi

doi:10.1002/sia.5522

Cited by 11 publications

(9 citation statements)

References 30 publications

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“…Therefore, single-cell MALDI-IMS analysis is a useful method for demonstrating lipid profiles and detecting rare target populations in human clinical samples. We suppose that the following methodological advances in the field of IMS made it possible to analyze such small objects: (1) the combined use of a microscope together with IMS and an improved laser ablation area to increase the spatial resolution up to 5 μm [27,31]; (2) the use of DHAP as a matrix for improved sensitivity of phospholipid detection by MALDI-IMS [21]; and (3) the sample preparation strategies such as the introduction of glutaraldehyde for cell fixation in IMS analysis [20].…”

Section: Discussionmentioning

confidence: 99%

“…A 12-well flexiPERM ® plate (Sarstedt, Tokyo, Japan) was affixed to the slide; MM cells and NPCs were applied into each well at a density of 1 × 10 4 cells/well, and cytocentrifuged using CytoSpin™ 4 (Thermo Scientific, Waltham, MA, USA) to spin the cells onto the surface of the glass slide. Cells were fixed with 100 μl of 0.25 % glutaraldehyde for 15 min and rinsed three times with 150 mM ammonium acetate buffer (pH 7.5) [20]. Samples were dried using an air blower and stored at −80°C until use.…”

Section: Ito Slide Preparationmentioning

confidence: 99%

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Decreased level of phosphatidylcholine (16:0/20:4) in multiple myeloma cells compared to plasma cells: a single-cell MALDI–IMS approach

et al. 2015

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Lipid metabolic changes under diseased conditions, particularly in solid tumors, are attracting increased attention. However, in non-solid tumors, including most hematopoietic tumors, lipid analyses are scarce. Multiple myeloma (MM) is a plasma cell disorder arising from bone marrow, and the lipid status of MM cells has not been reported yet. In this study, we analyzed flow cytometry-sorted single MM cells and normal plasma cells (NPCs) using matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS), a two-dimensional label-free mass spectrometry technique for biomolecular analysis, to obtain specific lipid information. We isolated 1.31-5.77% of MM cells and 0.03-0.24% of NPCs using fluorescence-activated cell sorting (FACS). Analysis of purified cells using MALDI-IMS at the single-cell level revealed that the peak intensity and ion signals of phosphatidylcholine [PC (16:0/20:4) + H](+) at m/z 782.5 were significantly decreased in MM cells compared to NPCs. By examining particular cell populations rather than cell mixtures, our method can become a suitable tool for the analysis of rare cell populations at the single-cell level and advance the understanding of MM progression.

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Section: Discussionmentioning

confidence: 99%

Section: Ito Slide Preparationmentioning

confidence: 99%

Decreased level of phosphatidylcholine (16:0/20:4) in multiple myeloma cells compared to plasma cells: a single-cell MALDI–IMS approach

et al. 2015

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“…29 Instead systematic investigations, especially those with a focus on lipids, still rely on other methods like glutaraldehyde fixation 30,31 or gelatin embedding. 9 In 2009, Malm et al compared cryofixation to chemical fixation via glutaraldehyde and freeze-drying (FD) to alcohol drying.…”

Section: A Sample Preparation Techniques For Cellsmentioning

confidence: 99%

“…17,18,26,31,33 Usually, only some lipid fragments are considered 17,26,33 or the investigation was carried out only in the positive ion mode, 26,33 while important lipid fragments such as fatty acids can only be seen in the negative ion mode. 18 Published comparisons are not comprehensive, e.g., Robinson and Castner 18 only analyzed formalin fixed cells; fixation via glutaraldehyde and secondary fixation via osmium tetroxide was not considered, and parameters of the freeze-drying procedure remain unclear.…”

Section: A Sample Preparation Techniques For Cellsmentioning

confidence: 99%

Assessment of different sample preparation routes for mass spectrometric monitoring and imaging of lipids in bone cells via ToF-SIMS

et al. 2015

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In ToF-SIMS analysis, the experimental outcome from cell experiments is to a great extent influenced by the sample preparation routine. In order to better judge this critical influence in the case of lipid analysis, a detailed comparison of different sample preparation routines is performed—aiming at an optimized preparation routine for systematic lipid imaging of cell cultures. For this purpose, human mesenchymal stem cells were analyzed: (a) as chemically fixed, (b) freeze-dried, and (c) frozen-hydrated. For chemical fixation, different fixatives, i.e., glutaraldehyde, paraformaldehyde, and a mixture of both, were tested with different postfixative handling procedures like storage in phosphate buffered saline, water or critical point drying. Furthermore, secondary lipid fixation via osmium tetroxide was taken into account and the effect of an ascending alcohol series with and without this secondary lipid fixation was evaluated. Concerning freeze-drying, three different postprocessing possibilities were examined. One can be considered as a pure cryofixation technique while the other two routes were based on chemical fixation. Cryofixation methods known from literature, i.e., freeze-fracturing and simple frozen-hydrated preparation, were also evaluated to complete the comparison of sample preparation techniques. Subsequent data evaluation of SIMS spectra in both, positive and negative, ion mode was performed via principal component analysis by use of peak sets representative for lipids. For freeze-fracturing, these experiments revealed poor reproducibility making this preparation route unsuitable for systematic investigations and statistic data evaluation. Freeze-drying after cryofixation showed improved reproducibility and well preserved lipid contents while the other freeze-drying procedures showed drawbacks in one of these criteria. In comparison, chemical fixation techniques via glutar- and/or paraformaldehyde proved most suitable in terms of reproducibility and preserved lipid contents, while alcohol and osmium treatment led to the extraction of lipids and are therefore not recommended.

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“…Moreover, it is well-known that sample storage is required during clinical studies because patient samples are usually not provided at the same time. 17 Finally, sample storage is crucial when experiments have to be repeated or verified or when samples are shipped between different laboratories.…”

Section: Introductionmentioning

confidence: 99%

Storage of cell samples for ToF-SIMS experiments—How to maintain sample integrity

et al. 2016

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In order to obtain comparable and reproducible results from time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis of biological cells, the influence of sample preparation and storage has to be carefully considered. It has been previously shown that the impact of the chosen preparation routine is crucial. In continuation of this work, the impact of storage needs to be addressed, as besides the fact that degradation will unavoidably take place, the effects of different storage procedures in combination with specific sample preparations remain largely unknown. Therefore, this work examines different wet (buffer, water, and alcohol) and dry (air-dried, freeze-dried, and critical-point-dried) storage procedures on human mesenchymal stem cell cultures. All cell samples were analyzed by ToF-SIMS immediately after preparation and after a storage period of 4 weeks. The obtained spectra were compared by principal component analysis with lipid- and amino acid-related signals known from the literature. In all dry storage procedures, notable degradation effects were observed, especially for lipid-, but also for amino acid-signal intensities. This leads to the conclusion that dried samples are to some extent easier to handle, yet the procedure is not the optimal storage solution. Degradation proceeds faster, which is possibly caused by oxidation reactions and cleaving enzymes that might still be active. Just as well, wet stored samples in alcohol struggle with decreased signal intensities from lipids and amino acids after storage. Compared to that, the wet stored samples in a buffered or pure aqueous environment revealed no degradation effects after 4 weeks. However, this storage bears a higher risk of fungi/bacterial contamination, as sterile conditions are typically not maintained. Thus, regular solution change is recommended for optimized storage conditions. Not directly exposing the samples to air, wet storage seems to minimize oxidation effects, and hence, buffer or water storage with regular renewal of the solution is recommended for short storage periods.

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Glutaraldehyde fixation method for single‐cell lipid analysis by time‐of‐flight secondary ion‐mass spectrometry

Cited by 11 publications

References 30 publications

Decreased level of phosphatidylcholine (16:0/20:4) in multiple myeloma cells compared to plasma cells: a single-cell MALDI–IMS approach

Decreased level of phosphatidylcholine (16:0/20:4) in multiple myeloma cells compared to plasma cells: a single-cell MALDI–IMS approach

Assessment of different sample preparation routes for mass spectrometric monitoring and imaging of lipids in bone cells via ToF-SIMS

Storage of cell samples for ToF-SIMS experiments—How to maintain sample integrity

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