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换一换Simple and fast technique for separating human mononuclear cells from small amounts of blood and buffy coat with high cell yield
Mononuclear cell separation
Magnetic beads
Genekam MNC isolator
Cell therapies
CAR-T
ELISA
B cells
T cells
CD4
CD8
Sudhir Bhatia, Gudrun Baersch
**We are again describing a rare and very simple protocol here:** The applications for human mononuclear cells (MNCs) are increasing, as these cells can be used for various tests, cell therapies and the development of human therapeutic antibodies. Density gradient isolation is still used today to separate MNCs. Diluted blood or buffy coat is added to the density gradient liquid, which is then centrifuged to obtain the different fractions. The white layer contains the MNCs, which are carefully pipetted out and washed to obtain the cells. The disadvantage of this laborious procedure is that blood and density gradient fluid may mix, which can lead to wastage of the valuable blood sample. We have developed a simple procedure in which the user waits a few minutes after mixing the blood sample in the Quick MNC isolator in a tube. The sample is then washed two or three times with PBS and centrifuged to obtain a pellet. Although small amounts of red blood cells occasionally remain in the pellet, they have no effect on the cells in the assay, e.g. when separating with magnetic beads, or on further experiments with isolated MNC cells. A high cell yield can be achieved with a single milliliter of blood or buffy coat. This MNC isolator method is an extremely fast, simple and effective method. Even very small amounts of buffy coat or blood, e.g. 100µl, can be used to isolate MNC. There is no documented case study in the literature using the density gradient method to separate MNC from such small amounts of blood or buffy coat. Our method is only option for small volumes. If the user cultivates the MNC in media with fetal calf serum (FCS), it is sufficient to use 2-3% FCS instead of the usual 10% FCS. This helps to reduce the financial burden on laboratories. Please read our publication in the reference section. Using this protocol, we have developed many cell cultures with MNC over the last 14 years.
AI 解读A versatile tissue-rolling technique for spatial-omics analyses of the entire murine gastrointestinal tract
Gustavo Monasterio, Rodrigo A. Morales, David A. Bejarano, Xesús M. Abalo, Jennifer Fransson, Ludvig Larsson, Andreas Schlitzer, Joakim Lundeberg, Srustidhar Das, Eduardo J. Villablanca
Tissues are dynamic and complex biological systems composed of specialized cell types that interact with each other for proper biological function. To comprehensively characterize and understand the cell circuitry underlying biological processes within tissues, it is crucial to preserve their spatial information. Here we report a simple mounting technique to maximize the area of the tissue to be analyzed, encompassing the whole length of the murine gastrointestinal (GI) tract, from mouth to rectum. Using this method, analysis of the whole murine GI tract can be performed in a single slide not only by means of histological staining, immunohistochemistry and in situ hybridization but also by multiplexed antibody staining and spatial transcriptomic approaches. We demonstrate the utility of our method in generating a comprehensive gene and protein expression profile of the whole GI tract by combining the versatile tissue-rolling technique with a cutting-edge transcriptomics method (Visium) and two cutting-edge proteomics methods (ChipCytometry and CODEX-PhenoCycler) in a systematic and easy-to-follow step-by-step procedure. The entire process, including tissue rolling, processing and sectioning, can be achieved within 2–3 d for all three methods. For Visium spatial transcriptomics, an additional 2 d are needed, whereas for spatial proteomics assays (ChipCytometry and CODEX-PhenoCycler), another 3–4 d might be considered. The whole process can be accomplished by researchers with skills in performing murine surgery, and standard histological and molecular biology methods.
AI 解读Single nuclei RNA sequencing (snRNA-seq) of frozen human lung tissue and hPCLS
Sequencing
Gene expression
Library Construction
Fragmentation
Ligation
snRNAseq
Lung
Senescence
SenNet
TriState
Single nuclei
PCLS
Frozen tissue
scRNAseq
Heidi Monroe, Nayra Cardenes, Oliver Eickelberg, Melanie Königshoff, Melanie Königshoff, Robert Lafyatis
Single-cell RNA sequencing (scRNA-seq) has become an essential tool for delineating cellular diversity in normal tissues and alterations in disease states. This technique requires the dissociation of tissue specimens into cell suspensions. However, the isolation of intact cells can be challenging due to factors such as fragility, large size and tight interconnections. Additionally, single-nuclei isolation can be performed on frozen tissue, enabling the analysis of biobanked samples in a single batch. This protocol for single-nuclei RNA sequencing (snRNA-seq) provides an alternative approach to scRNA-seq, overcoming these limitations to generate high-quality transcriptomic data. The analysis of gene expression at the cellular level has proven to be a powerful tool for understanding various aspects of lung biology and disease, particularly the process of lung aging. Aging affects different cell types within the human lung heterogeneously, leading to a range of associated changes in their function. Examining the patterns of gene expression in the numerous lung cell types provides insights into the aging and disease processes that contribute to cellular dysfunction. By analyzing these changes at the single-cell level, we can delineate the complex cellular diversity in the human lung and track alterations in molecular pathways involved in the dynamic process of lung aging. Understanding these gene expression patterns will offer opportunities for timely interventions and the identification of biomarker for early prognosis and personalized treatment therapies. This protocol collection describes the process of single nuclei RNA sequencing from nuclei isolation from frozen tissue (whole, or from PCLS), to barcoding, library construction and sequencing.
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