Cell Biology Typical parameters optimized during cell-based assay development are Cell density, viability of cells, reagent concentrations, incubation period, temperature, instrument settings, DMSO test (vehicle control) Method development, Plate uniformity Studies 3 plates are run on three consecutive days: HML, LHM, MLH format
Test-Retest for Mean Statistics Ratio On two consecutive days, 10-20 compounds are run with varying potencies in CRC to get the MSR (mean statistics ratio) Target based cell-based assays by Fluorescence, Luminescence & GPCR targets-based assays using transfected cells Assay Development and Validation – In-vitro Biochemical and Cell based assays are validated as per NCGC guidelines Parameters optimized during biochemical assay development include – Km determination for the substrate with fixed enzyme concentration Enzyme titration with fixed substrate concentration to choose optimal enzyme concentration with good Vmax Reaction time course studies to evaluate the linearity of the reaction DMSO toleration studies to maintain constant concentration in the assay Enzyme stability test under varying pH conditions and optimize ideal pH range Optimization of pre-incubation time for screening of compounds Once the assay conditions are optimized, the assay will be validated with known reference compounds minimum 10 Cell Based Assays and Bioassays Cell based ELISA assays, Flow cytometer studies, Apoptotic, FRET, TRF, HTRF, Kinetic Assays, Cell proliferation studies Biomarker samples ELISA Analysis – Leptin, Lipoprotein A, Ghrelin, Free fatty acids, TNfα, IL6, Adiponectin, HSP90, HSP27, Insulin, etc. Primary cells-based assays- Keratinocytes, epithelial, blood brain barrier micro vessel cells, hepatocytes, lymphocytes, Mast cells Mitochondrial membrane potential assay, DNA ladder assay, Clonogenic assay, Hemolysis assay Cell based assays for Cancer (100 cancer cell lines from ATCC, USA), Diabetes (10 cell lines), AD (5 cell lines), Inflammation (10 cell lines), Osteoporosis (5 cell lines) Flow Cytometry Assays, Gene expression, Western Blotting and Cell ELISA, Comet assay, DNA fragmentation, Whole Blood Assays, Cell Viability (LDH, MTT, Alamar Blue), TUNEL assay Anti-inflammatory cell-based assays, Osteoporosis assays – Osteoblast (UMR 106 cells) & Osteoclast (MG63 cells), Immunofluorescence based assays, Macrophages assay, Antioxidants assays Insulinogenic activity of test compounds using RIN m5F cells, MIN6, Glucose uptake assay using 3T3L1 adipocytes CAMP assay in UMR106 cells (Osteoblast), MG63 (Osteoclast cells), Inflammatory assays, Macrophages assay, Cell based antioxidants
