immunomagnetic cd45 depletion easysep human cd45 depletion kit ii  (STEMCELL Technologies Inc)

Journal: bioRxiv

Article Title: Mapping the Cellular Biogeography of Human Bone Marrow Niches Using Single-Cell Transcriptomics and Proteomic Imaging

doi: 10.1101/2024.03.14.585083

Figure Lengend Snippet: A ) UMAP computed from 19,257 mesenchymal cells from 12 individuals showing different mesenchymal subsets, with RNAlo MSCs being excluded. B ) Dot plot showing normalized expression of key mesenchymal marker genes in MSC subsets. Rows and columns were manually ordered. C ) Dot plot showing the normalized expression of literature-derived marker genes for human MSCs including NT5E (CD73), THY1 (CD90), and ENG (CD105). NGFR (CD271) and MCAM (CD146) have also been described as canonical MSC markers. D ) CytoTRACE analysis projected onto the MSC UMAP showing the predicted differentiation score, where higher values imply the cell is more primitive. E) Boxplots showing the relative colony forming potential (number of colonies produced by each cell type divided by total colonies from each sample, each data point is one sample) of sorted Fibro-MSC (CD45-CD38-CD235ab-VECAD-PDPN+), THY1+ MSC (CD45-CD38-CD235ab-VECAD-PDPN-LEPR+ CD90+), Adipo-MSC (CD45-CD38-CD235ab-VECAD-PDPN-LEPR+ CD90-), and osteolineage cells (CD45-CD38-CD235ab-VECAD-PDPN-CD56+). P-values were computed as Fibro-MSC vs. all by Dunnett’s Multiple Comparisons test. F) Line plot showing the population doublings over the course of cell culturing for eight passages, where all cells were passaged every 7 days. P-values are computed as Fibro-MSC vs other MSCs by two-way ANOVA. G ) UMAP showing 3,874 endothelial cells from the 12 individuals. SEC – Sinusoidal Endothelial Cells, AEC – Arterial Endothelial Cells. H ) Dot plot showing normalized expression of top differentially expressed genes by adjusted p-value between endothelial subsets, as well as CDH5 , CD34 , and KDR which were manually selected as pan-endothelial markers. I ) MSCs from human bone marrow aspirate reported from De Jong et al., Nature Immunology , 2021 were reference mapped to our scRNA-Seq atlas and the cell type labels were predicted. J ) Human fetal bone marrow mesenchymal cells from Jardine et al., Nature , 2021 were reference mapped to our scRNA-Seq atlas and the cell labels were predicted.

Article Snippet: 20uL of RBC depleted cells were put on ice, while 67% of the remaining cells were used for immunomagnetic CD45 depletion using EasySep Human CD45 Depletion Kit II (STEMCELL # 17898) to isolate non-hematopoietic cells in an unbiased fashion, while 33% of cells were subjected to immunomagnetic CD34 enrichment using EasySep Human CD34 Positive Selection Kit II (STEMCELL #17856).

Techniques: Expressing, Marker, Derivative Assay, Produced, Cell Culture