Analytical Core B Abstract The Analytical Core (Core B) will be organized to provide high quality, reproducible, low cost, automated high- throughput robotic analytical services to the three Projects. The Core will be managed and staffed with experts who have demonstrated an ability to develop, engineer, and implement the assays necessary to support the proposed activities of the Analytical Core. The Analytical Core will be organized around 5 major services/specific aims.
Specific Aim 1 : To provide high quality, reproducible automated assays to support Projects 1-3.
Specific Aim 2 : To provide genotyping services for Projects 1 and 2. We will test for the presence of single nucleotide polymorphisms (SNPs) in the subjects recruited under Project 2.
Specific Aim 3 : To provide human renal proximal tubule (RPT) cells as well as renal distal tubule (RDT) and renal collecting duct (RCD) cells for Projects 1 and 3. We will also isolate, immortalize and culture RPT, RTD and RCD cells from fresh human urine specimens from Project 2. Mouse urine cells will be obtained from Project 3.
Specific Aim 4 : To provide cellular imaging services to support Projects 1 and 3. In addition, in support of Project 2, we will study cells from the urine of subjects treated as outlined in the protocols of Project 2. We will also study cells isolated from mouse urine obtained from Project 3.
Specific Aim 5 : To provide flow cytometry services to support Projects 1-3. 1. Robotic Analytical Platforms High Throughput Assays High Content Assays 5. Flow Cytometry Accuri Urine Cell Assays Analytical Core B 2. Molecular Biology: SNPs for salt sensitivity and hypertension DNA extraction and Biobanking 4. Confocal Microscopy: FRET pH Assays Cytoplasmic cAMP, cGMP sensors Intracellular Na+ fluorescence 3. Cell Culture: RPT, RDT, RCD cell lines from human kidney tissue and urine Microcarrier-3D culture
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