Apoptosis (Live cell assay) : Both 2D and 3D models : Our live cell assay measures the ability of the drug to induce caspase activation in cancer cells using stable cells expressing FRET probe for activated caspases. We have cells engineered with a FRET probe in the nucleus so that automated segmentation and analysis is quite easy to perform using high-throughput imagers. The following validated cell lines are available with stable expression of FRET probe NLS. (More will be included in the list soon).
The following validated cell lines are available with stable expression of FRET probe NLS. (More will be included in the list soon).
Breast cancer: MCF-7, T47D
Colon cancer: SW480, HCT 116, SW620
Cervical Cancer: SiHa, HeLa
Osteosarcoma: U205
Neuroblastoma: U251
Ovarian Cancer: OVCAR8, NCI Adr.Res
Pancreatic cancer (MiaPaca2, PANC-1)
(Any relevant three cells may be ideal for initial screening).

Assays for Autophagy and screening of m-TOR pathway inhibitors: Induction of autophagy and inhibitors of m-TOR pathway is also emerging as an important target in cancer. We have generated stable cells expressing LC3 EGFP and its translocation to autophagic vacuoles is used as the readout by imaging approaches and is adapted for high-throughput image based drug screening.
Available cell lines: OVCAR8, HCT116, DLD

Mitophagy inducers or inhibitor screening: The strategy employs high-throughput image based approach using stable cancer cell lines expressing sensors of Mitophagy.

Angiogenesis

Method1: We have immortalized human micro- vascular endothelial cells that form quantifiable angiogenic tubules on matrigel. Hence inhibition of tubule formation can be used as the read out for activity. Since the cell line is immortalized, available for screening of large number of compounds using an image based system is feasible.

Method2: Rat aortic ring assay: Dorsal aortic rings will be prepared from rat aorta will be placed in a collagen pre-coated 96-well plates and treated with compounds of interest. Rings will be analysed through phase-contrast microscopy to study microvessel out growth.

Assay for antioxidant potential using mammalian cells: Nuclear factor erythroid 2-related factor 2 (Nrf2) is the master transcription factor of the antioxidant response element pathway, coordinating the induction of detoxifying and antioxidant enzymes. Nrf2 is normally sequestered in the cytoplasm by Kelch-like ECH-associating protein 1 (Keap1) and upon activation tans ocate to nucleus. We have developed NRF2 EGFP stable cells so that nuclear translocation can be used as a platform for initial screening. An approach for identification of strong antioxidants that break the interaction is also available and utilizes FRET approach.

Endoplasmic reticulum calcium releasing compounds: We also developed high throughput calcium imaging assay based on a FRET-based calcium indicator at single cell resolution for compound screening.

Tumor stem cell assays:Tumor stem cells contribute both for tumor initiation and tumor drug resistance forming an ideal target in cancer. However, they are extremely rare in cancer cells and so difficult to be used for drug screening. We have designed several assays to study the effect of compounds on tumor stem cells. From established cell cultures and surgically dissected tumor samples, we have optimized conditions to isolate stem cell like cells by FACS which can be used for identifying compounds that specifically target tumor stem cells using 2D and 3D culture system The spheroid formation assay can also be used to study cancer stem cells. Here cells will be plated in low attachment plates and then treated with compounds of interest. Cells will be observed under phase-contrast microscope to analyse spheroid formation. Stem cells will form spheroids under these conditions and effect of compounds on spheroid formation will be analysed.