FixNCut: single-cell genomics through reversible tissue fixation and dissociation.

The use of single-cell technologies for clinical applications requires disconnecting sampling from downstream processing steps. Early sample preservation can further increase robustness and reproducibility by avoiding artifacts introduced during specimen handling. We present FixNCut, a methodology for the reversible fixation of tissue followed by dissociation that overcomes current limitations. We applied FixNCut to human and mouse tissues to demonstrate the preservation of RNA integrity, sequencing library complexity, and cellular composition, while diminishing stress-related artifacts. Besides single-cell RNA sequencing, FixNCut is compatible with multiple single-cell and spatial technologies, making it a versatile tool for robust and flexible study designs.

Comparative flow cytometry-based immunophenotyping analysis of peripheral blood leukocytes before and after fixation with paraformaldehyde.

Flow cytometry based immunophenotyping provides prime insight into cellular population composition and characteristics, and is widely used in basic and clinical research. Challenges in processing peripheral blood samples in a timely manner necessitate protocol adaptations and utilization of fixatives. Fixation, however, may introduce artifacts to the flow cytometry readout. We performed a comparative flow cytometry immunophenotyping analysis of 13 immune cell populations in the whole blood using a staining protocol with and without fixation step. Freshly procured human peripheral blood samples were stained with a panel of 33 fluorochrome-conjugated antibodies. Samples were processed using a protocol with or without a paraformaldehyde-based fixation step, and matching sample pairs were analyzed by flow cytometry. Our results show that paraformaldehyde-based fixation, in comparison to matched unfixed samples, did not significantly affect population distribution and frequency for: B cells, Plasmablasts, Dendritic cells, NK cells, Granulocytes, Neutrophils, Eosinophils, or Hematopoietic Stem/Progenitor Cells. However, fixation led to significant marker shifts in the subpopulation distribution in CD4, T regulatory, CD8, Monocytes, and Basophils. These results indicate the importance of pre-experimental assessment of fixation-introduced artifacts in the flow cytometry output when considering the feasibility of fresh processing. This is especially important for samples analyzed using comprehensive exploratory immunoprofiling panels.

Impact of blood storage and sample handling on quality of high dimensional flow cytometric data in multicenter clinical research.

Obtaining reliable and reproducible high quality data in multicenter clinical research settings requires design of optimal standard operating procedures. While the need for standardization in sample processing and data analysis is well-recognized, the impact of sample handling in the pre-analytical phase remains underestimated. We evaluated the impact of sample storage time (≈transport time) and temperature, type of anticoagulant, and limited blood volume on reproducibility of flow cytometric studies. EDTA and Na-Heparin samples processed with the EuroFlow bulk lysis protocol, stained and stored at 4 °C showed fairly stable expression of cell surface markers and distribution of the major leukocyte populations for up to 72 h. Additional sample fixation (1% PFA, Fix & Perm) did not have any beneficial effects. Blood samples stored for <24 h at room temperature before processing and staining seemed suitable for reliable immunophenotyping, although losses in absolute cell numbers were observed. The major losses were observed in myeloid cells and monocytes, while lymphocytes seemed less affected. Expression of cell surface markers and population distribution were more stable in Na-Heparin blood than in EDTA blood. However, storage of Na-Heparin samples was associated with faster decrease in leukocyte counts over time. Whole blood fixation strategies (Cyto-Chex, TransFix) improved long-term population distribution, but were detrimental for expression of cellular markers. The main conclusions from this study on healthy donor blood samples were successfully confirmed in EDTA clinical (patient) blood samples with different time delays until processing. Finally, we recognized the need for adjustments in bulk lysis in case of insufficient blood volumes. Despite clear overall conclusions, individual markers and cell populations had different preferred conditions. Therefore, specific guidelines for sample handling should always be adjusted to the clinical application and the main target leukocyte population.

Preparation of Plant Material for Analysis of Protein-Nucleic Acid Interactions by FRET-FLIM.

DNA-binding proteins are involved in the dynamic regulation of various cellular processes such as recombination, replication, and transcription. For investigating dynamic assembly and disassembly of molecular complexes in living cells, fluorescence microscopy represents a tremendous tool in biology. A fluorescence resonance energy transfer (FRET) approach coupled to fluorescence lifetime imaging microscopy (FLIM) has been used recently to monitor protein-DNA associations in plant cells. With this approach, the donor fluorophore is a GFP-tagged binding partner expressed in plant cells. A Sytox® Orange treatment converts nuclear nucleic acids to FRET acceptors. A decrease of GFP lifetime is due to FRET between donor and acceptor, indicating close association of the GFP binding partner and Sytox® Orange-stained DNA. In this chapter, we present a step-by-step protocol for the transient expression in N. benthamiana of GFP-tagged proteins and the fixation and permeabilization procedures used for the preparation of plant material aimed at detecting protein-nucleic acid interactions by FRET-FLIM measurements.

Multi-element analysis of size-segregated fine and ultrafine particulate via Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry.

In this study a novel and reliable Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS) measurement protocol for the elemental characterization of size-segregated particulate was developed. Special efforts were made to improve and optimize sample pre-treatment steps and LA operating conditions to avoid some critical drawbacks encountered during analysis and to make the particulate samples suitable for an accurate and reproducible LA-ICP-MS analysis, regardless of the mass loading on each filter. For example, a new approach for dust-fixation on the sample-carrier was developed using a glycerol coverage, which allowed to overcome problematic sample losses during the ablation process. Under the optimum conditions, dust samples, blank filters and standards for calibration were analyzed by multiple rastering of defined spot areas. Quantitative analysis was accomplished with dried micro-droplets of aqueous standard solutions. Derived method detection limits varied between 0.001 and 0.1 ng m-3 and allowed even for the smallest particle fraction quantitative measurements. The accuracy of LA-ICP-MS results was verified by comparison with conventional ICP-MS analysis of selected PM samples after sample mineralization. The proposed LA treatment procedure benefits from a simple and fast sample preparation, thus overcoming the laborious pre-treatment steps required for wet chemical digestion. Moreover, the better sensitivity of the LA-ICP-MS approach provided more complete information about the mass concentration and size-distribution of the investigated elements, thus allowing to deeper investigate the composition of the most dangerous PM fractions in terms of health concern.

Purification and Application of a Small Actin Probe for Single-Molecule Localization Microscopy.

The cytoskeleton is involved in many cellular processes. Over the last decade, super-resolution microscopy has become widely available to image cytoskeletal structures, such as microtubules and actin, with great detail. For example, Single-Molecule Localization Microscopy (SMLM) achieves resolutions of 5-50 nm through repetitive sparse labeling of samples, followed by Point-Spread-Function analysis of individual fluorophores. Whereas initially this approach depended on the controlled photoswitching of fluorophores targeted to the structure of interest, alternative techniques now depend on the transient binding of fluorescently labeled probes, such as the small polypeptide lifeAct that can transiently interact with polymerized actin. These techniques allow for simple multicolor imaging and are no longer limited by a fluorophore's blinking properties. Here we describe a detailed step-by-step protocol to purify, label, and utilize the lifeAct fragment for SMLM. This purification and labeling strategy can potentially be extended to a variety of protein fragments compatible with SMLM.

Post-sampling release of free fatty acids - effects of heat stabilization and methods of euthanasia.

INTRODUCTION: The field of lipid research has made progress and it is now possible to study the lipidome of cells and organelles. A basic requirement of a successful lipid study is adequate pre-analytical sample handling, as some lipids can be unstable and postmortem changes can cause substantial accumulation of free fatty acids (FFAs). METHODS: The aim of the present study was to investigate the effects of conductive heat stabilization and euthanasia methods on FFA levels in the rat brain and liver using liquid chromatography tandem mass spectrometry. RESULTS: The analysis of brain homogenates clearly demonstrated phospholipase activity and time-dependent post-sampling changes in the lipid pool of snap frozen non-stabilized tissue. There was a significant increase in FFAs already at 2min, which continued over time. Heat stabilization was shown to be an efficient method to reduce phospholipase activity and ex vivo lipolysis. Post-sampling effects due to tissue thawing and sample preparation induced a massive release of FFAs (up to 3700%) from non-stabilized liver and brain tissues compared to heat stabilized tissue. Furthermore, the choice of euthanasia method significantly influenced the levels of FFAs in the brain. The FFAs were decreased by 15-44% in the group of animals euthanized by pentobarbital injection compared with CO2 inhalation or decapitation. DISCUSSION: Our results highlight the importance of considering euthanasia methods and pre-analytical treatment in lipid analysis, factors which may otherwise interfere with the outcome of the experiments.