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换一换Cell preparation for scRNA-Seq from diluted bodily fluids
Linas Mazutis, Vaidotas Kiseliovas, Adrienne Boire
Efficient isolation of cells from complex bodily fluids is a crucial step for many biological and biomedical applications. Yet, it can become a particularly challenging when only a small fraction of cells is dispersed in a large volume of fluid, e.g. ~ 1000-10000 cells / 10 mL. In such cases a conventional wisdom would suggest the use of canonical 15 or 50 ml tubes and centrifugation forces at 500-800g to pellet the cells. However, hard centrifugation can damage the cells and rupture their membrane, while at lower centrifugation speeds the sedimentation rates are slow and might be insufficient to form a cell pellet. Moreover, intrinsic variability of cells in terms of size (6-20 µm) and density (1.06-1.29g/mL) may reduce the pelleting and recovery yields. When using swinging bucket rotors and large sample volumes (> 5 mL) the sample sedimentation run times can become very long due to the increased sedimentation path length (distance that cells need to travel to reach bottom of the tube). On another hand, in fixed angle rotors the cells will travel only a short distance before hitting the wall of the tube and as a result will migrate down the wall forming a long trail until reaching the bottom of the tube, which will preclude formation of a tight pellet. Overall, when working with fragile cell samples preference should be given to lower centrifugal forces and shorter centrifugation times. These two requirements can be fulfilled by using smaller tubes as the shorter sedimentation path length will shorten the distance that cells need to travel to reach bottom of the tube. Given these considerations this protocol describes the isolation of viable primary cells from diluted suspensions of bodily fluids. The protocol exemplifies the isolation of cells (immune and cancer cells) from cerebrospinal fluid where the concentration of cells is in the order of 0-20 cells / µl.
AI 解读Chromium Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression (CG000505)
10x Genomics
The Chromium Nuclei Isolation Kit is an all-in-one solution for the standardized isolation of nuclei from frozen tissue for use in 10x Genomics Single Cell assays. Frozen tissue samples are homogenized with a pestle in Lysis Buffer and passed through a column. Next, debris is removed via centrifugation in Debris Removal Buffer. The isolated nuclei are then washed and resuspended and loaded directly into compatible 10x Genomics Single Cell assays. The Chromium Nuclei Isolation Kit streamlines the nuclei isolation process into a single workflow, allowing for increased efficiency, scalability through sample batching, and reduced experimental variability using 10x Genomics pre-formulated reagents. The protocol is designed to be compatible with a wide variety of tissue types and sizes. This User Guide outlines the process for isolating Nuclei from frozen tissues for use in compatible 10x Genomics Single Cell assays. Refer to the Product Compatibility and Protocol Selector pages for additional information on choosing the appropriate nuclei isolation kit and protocol based on the intended downstream Single Cell assay.
AI 解读Structural prediction of VPS13C with AlphaFold2
Shujun Cai, Pietro De Camilli
This protocol describes the procedure of structural prediction of full-length human VPS13C and its truncation mutant with AlphaFold2 and the procedure to combine each segments into one structure.
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