Test-Retest for Mean significance ratio
- On two consecutive days, 10-20 compounds are run with varying potencies in CRC to get the MSR (Mean significance ratio)
- Target based cell-based assays by Fluorescence, Luminescence & GPCR targets-based assays using transfected cells
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
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
