Project description:The goal of this study was to test if lipid-based cell hashing worked in salamander species and to test how lipid-based hashing compared to demultiplexing samples based on species origin and single nucleotide polymorphisms (SNP). Spleens were taken from four animals in total: one adult Pleurodeles waltl (female, 23.5grams and 16.1cm snout-to-tail, strain: tgSceI(CAG:loxP-GFP-loxP-Cherry)Simon), one adult Pleurodeles waltl (male, 13.95g and 15.7cm, tgSceI(CAG:loxP-GFP-loxP-Cherry)Simon), one adult Notophthalmus (female, 4.45g and 10.6cm, WT), and one adult Notophthalmus (male, 3.55g and 10.2cm, WT). Samples were stained with CM304 (Pleurodeles female), CMO305 (Pleurodeles male), and CMO306 (pool of both Notophthalmus samples) as per 10x Genomics 3’ Cellplex labeling protocol (Demonstrated Protocol, CG000391). These samples were processed through the 10x Controller and with Cellranger 7.0 using a dual species reference. The sample origin was determined by lipid-based hashing and then compared to mapping rates to each species transcriptome and using SNP-based demultiplexing.
Project description:The exit of antigen-presenting cells (APC) and lymphocytes from inflamed skin to afferent lymph is vital for the initiation and maintenance of dermal immune responses. How such exit is achieved and how cells transmigrate the distinct endothelium of lymphatic vessels is however unknown. Here we show that inflammatory cytokines trigger activation of dermal lymphatic endothelial cells (LEC) leading to expression of the key leukocyte adhesion receptors ICAM-1, VCAM-1 and E-selectin, as well as a discrete panel of chemokines and other potential regulators of leukocyte transmigration. Furthermore, we show that both ICAM-1 and VCAM-1 are induced in the dermal lymphatic vessels of mice exposed to skin contact hypersensitivity where they mediate lymph node trafficking of DC via afferent lymphatics. Lastly, we show that TNF_-stimulates both DC adhesion and transmigration of dermal LEC monolayers in vitro and that the process is efficiently inhibited by ICAM-1 and VCAM-1 adhesion-blocking mAbs. These results reveal a CAM-mediated mechanism for recruiting leukocytes to the lymph nodes in inflammation and highlight the process of lymphatic transmigration as a potential new target for anti-inflammatory therapy. Experiment Overall Design: Global gene expression profile of normal dermal lymphatic endothelial cells cultured in media alone (no TNF) compared to that of normal dermal lymphatic endothelial cells stimulated with TNFalpha, 1 ng/ml for 48h.Triplicate biological samples were analyzed from human lymphatic endothelial cells (3 x controls; 3 x TNF treated) and a single sample analyzed from mouse lymphatic endothelial cells (1 x controls; 1 x TNF treated).
Project description:Comparison of the transcriptome of immortalised mouse kidney epithelial cells either wt for Fh1 or Fh1-deficient. The cells were isolated from kidneys of P5 mouse(see Frezza et al, Nature 2011). Briefly, Fh1_fl (flox) are wt for Fh1 (floxed cassette not excised), clone 1 and clone 19 are two different Fh1-deificent clones (floxed cassette excised) and Rec are clone 19 with reconstituted Fh1 expression from exogenous plasmid.
Project description:The long term objective is to create an encyclopedia of the expression levels of all genes in multiple components of the developing kidney. The central thesis is straightforward. The combination of fluorescent activated cell sorting (FACS) plus microarray analysis offers a powerful, efficient and effective method for the creation of a global gene expression atlas of the developing kidney. Microarrays with essentially complete genome coverage can be used to quantitate expression levels of every gene in FACS isolated components of the developing kidney. The ensuing rapid read-out provides an expression atlas that is more sensitive, more economical and more complete than would be possible by in situ hybridizations alone. MafB-GFP BAC transgenic mice were utilized to isolate the podocyte cells from the developing embryonic kidneys. Podocyte cells were isolated from the kidney using trypsinization and FACS. RNA was isolated and the gene expression profiles were determined by microarrays.
Project description:The long term objective is to create an encyclopedia of the expression levels of all genes in multiple components of the developing kidney. The central thesis is straightforward. The combination of fluorescent activated cell sorting (FACS) plus microarray analysis offers a powerful, efficient and effective method for the creation of a global gene expression atlas of the developing kidney. Microarrays with essentially complete genome coverage can be used to quantitate expression levels of every gene in FACS isolated components of the developing kidney. The ensuing rapid read-out provides an expression atlas that is more sensitive, more economical and more complete than would be possible by in situ hybridizations alone Renin-GFP transgenic mice were utilized to isolate renin producing cells from the kidneys of either P0 neonatal pups or Adult mice. Renin producing cells were isolated from the kidney by fluorescent activated cell sorting (FACS). RNA was isolated from FACS sorted cells and the gene expression profiles were determined by microarrays.
Project description:T cells (all CD4 or Treg) measured by RNA-seq, infected or not-infected by N.brasiliensis, across different tissues, with or without CD4-Cre RORA KO. Several different cohorts were analyzed, either as a time course (TC), two rounds of analyzing WT vs RORA KO differences (oldko and newko), and a comparison of CD4+Foxp3+ Tregs specifically (tregkovswt)