In-Vitro Biology

Molecular Biology

SKANDA provides a wide range of services in the area of recombinant DNA technology, strain engineering and gene expression analysis.

Gene Expression

Expression of a targeted gene at the transcriptional and translational levels in host is important in studying the functional gene. Scientists at routinely carry out the following gene expression analysis:

• Real Time PCR
• Western blotting
• ELISA
• Flow Cytometry
• Mass spectrometry
• RNA sequencing

Molecular Biology

• Automated DNA sequencer- ABI 3730 XL, ABI 3130XL
• Thermal Cyclers
• Real-Time PCR instruments (Biorad & ABI)
• Pulsed field electrophoresis
• Gel Documentation Systems
• Nano Drop Spectrophotometer

Microbiology

• Bench Top Fermenters
• Biosafety Cabinets- BSL I & II
• Incubators and Incubator Shakers

 Services

• Gene expression studies by PCR, RT-PCR and qPCR methods.
• DNA cloning, DNA sequencing, gene expression and western blotting services.
• Cancer gene expression study Caspase3, Caspase8, Caspase9 BCL2, Bax, Brac 1, Brac 2, HSP90 and HSP27.
• RAPD, RFLP, Next generation sequencing- metagenomics, whole genome sequencing, Transcriptome analysis
• Transfection of clones in mammalian cell lines CHO-K1, HEK 293, 3T3, MCF7 etc.
• Clonal selection, amplification, analysis of expression by various Biochemicals and functional methods.
• Native PAGE, SDS, PAGE, 2D electrophoresis, Protein expression
• Molecular identification of bacteria, fungi – 16s and 18s rRNA sequencing and phylogenic analysis.

Antibody Production & Purification

Skanda is a preferred partner for antibody production, we leverage the scientific expertise and flexibility for high quality Mab. Our experienced scientists offer small and large scale monoclonal and polyclonal antibody production and purification. We also offer hybridoma generation and screening services.

Polyclonal

• Polyclonal antibodies production – New Zealand White Rabbit standard or custom requirement
• Variety of antigens (bacterial, fusion proteins & peptides)

Hybridoma

• Immunization of mice/rat
• Conjugation for immunization
• Animal care & handling
• Hybridoma generation and screening by ELISA and Biacore
• Screening for biological (neutralization, internalization etc.)
• Titer determination, Sub cloning and isotyping of selected clones
• Purification and analysis of monoclonal antibodies
• Cryopreservation of cell lines

Recombinant MAbs

• Transient transfections (in HEK293) and/ or stable cell line generation (in CHO-based expression systems)
• Cloning of antibody genes from hybridoma and generation of high-expressing plasmid constructs
• Production from small scale to large scale cultures
• Purification of antibodies, Cell banking and quality control

Microbiology Studies

Microbial threat and the impending resistance to existing antibacterial agents has led to development of next-generation broad spectrum antibiotics to fight the most antibiotic resistant strains.

• Antimicrobial studies for NCE – Antibacterial, Antifungal activity against various pathogens
• Isolation & characterization microbes from various sources —Clinical, Environmental, metagenomics
• Detection of Pathogens as per WHO & FIP ( coli, Salmonella, Pseudomonas, Klebsiella pneumonia, Staphylococcus aureus, streptococcus sp)
• Residual Anti-Microbial Substances
• Bacterial Enterotoxins, Mycotoxins
• AMES Mutagenicity, Sterility and LAL test
• Disinfection Efficacy & Preservative Efficacy, Potency of Antibiotics and Vitamins
• Minimum inhibitory concentration determination by micro broth dilution as per CLSI standards

Technology platforms & infrastructure

• Binding (radio ligand, SPA) — Trilux, Top count
• HTRF, TR-FRET, Alpha ELISA, Florescence, Luminescence — Molecular Devices I3X (6well – 1536 well plate), Tecan, High Content Imager (Cellomics)
• Immunological assays — MSD platform (multiplexing), ELISA
• Cell count, Cell cycle, Cell surface markers — FACS Dual calibur (4-channel), Florescence Microscope & Differential counter
• Gene Expression & protein work. — PCR, qRT-PCR system, Taqman, Western blot – Chemidoc
• Direct analyte measurement —- HPLC, LCMS/MS, GCMS/MS

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

 Assay Development and Validation; Cell based assays

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)

Cell Based Assays and Bioassays

• Target based cell-based assays by Fluorescence, Luminescence & GPCR targets-based assays using transfected cells
• 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

Our CPCSEA recognized in vivo facility houses 25 rooms with animal maintenance as per the international standards. With a broad-spectrum of studies on various animal models such as Rats, Mice, Rabbits, Guinea Pigs, & Hamsters the experts at Skanda Life Sciences provides most time-efficient results at a cost-effective pricing with successful execution and completion of study plans on a global level. Some of the most recognized and efficacious study topics of the organization are listed as:

• Pharmacological studies
• ADME/DMPK studies
• Efficacy model studies
• Virology studies

Anti-Viral Study

• In vitro and In vivo assessment of Immunomodulatory effects against HCoV-229E, H1N1, Zika Virus,
• Cytokine levels –IgG, IgM, IL-6, IFN-γ, and TNF-α, IL-2, IL-12, IL-10
• Immuno-phenotyping of cells by flowcytometry –T cells, B cells, NK cells
• Haematological, biochemical and Histopathology of organs
• In-vitro neutralization assay
• In-vitro immunogenicity using PBMCs
• Cytotoxic evaluation of novel anti-viral peptide in VERO cells
• Plaque Reduction Assay
• In vivo efficacy of novel anti-viral

Effect of novel anti-viral peptide in preventing the viral protein synthesis