This document provides a list of 10 core assays for testing compounds that modulate the immune system. The assays measure things like cell proliferation, nitric oxide release from macrophages, and cytokine release from splenocytes or human PBMCs in response to test compounds. Each assay is described in terms of the test model, turnaround time, minimum compound requirement, and standard reporting format. The assays will help optimize leads for anti-inflammatory, pro-inflammatory, or other immunomodulatory activities.

1. IN-VITRO CELL BASED ASSAY- CORE ASSAY LIST 2011

2. CORE ASSAY LIST 2011
THERAPEUTIC AREA – IMMUNOMODULATION
Turn Minimum
Project
REF# Assay Principle Around Compound Standard Format
Type
Time Required
Test model: Determine effect of mitogens/inhibitors on cell
proliferation in murine splenocytes/Human PBMC. Useful for lead Up to 3 compounds at
Cell
CA01 optimization of proliferation/ anti-proliferation compounds. 4 Weeks 10 mg 5 concentrations tested
Proliferation
Assay format: Incubations in 48-well plates. MTT read out. in quadruplicate
Data format: WordTM report with data available in an ExcelTM file.
Test model: Determine effect of compounds on (un-stimulated)
Nitric Oxide release from murine macrophages. Useful for lead
Up to 4 compounds at
Mediator optimization for pro-inflammatory compounds and potency
CA02 4 Weeks 10 mg 5 concentrations tested
release determination.
in triplicate
Assay format: Incubations in 96-well plates. Colorimetric.
Data format: WordTM report with data available in an ExcelTM file.
Test model: Determine effect of compounds on (stimulated)
cytokine release (IL-2) from murine splenocytes. Useful for lead
Up to 3 compounds at
Mediator optimization for pro-inflammatory compounds and potency
CA03 4 Weeks 10 mg 5 concentrations tested
release determination.
in quadruplicate
Assay format: Incubations in 48-well plates. ELISA.
Data format: WordTM report with data available in an ExcelTM file.
Test model: Determine effect of compounds on (stimulated)
cytokine release (IL-3) from murine splenocytes. Useful for lead
Up to 3 compounds at
Mediator optimization for pro-inflammatory compounds and potency
CA04 4 Weeks 10 mg 5 concentrations tested
release determination.
in quadruplicate
Assay format: Incubations in 48-well plates. ELISA.
Data format: WordTM report with data available in an ExcelTM file.
R & D Centre: Plot # 5B, 19th K.M Stone, Hosur Road, Bangalore – 560 100, India.
Tel: 91-080-7832265, 7833092, 7834872, Fax: 91-080-7834369, E-mail: cvc@naturalremedy.com

3. THERAPEUTIC AREA – IMMUNOMODULATION
Turn Minimum
Project
REF# Assay Principle Around Compound Standard Format
Type
Time Required
Test model: Determine effect of compounds on (stimulated)
cytokine release (IL-4) from murine splenocytes. Useful for lead
Up to 3 compounds at
Mediator optimization for Anti-inflammatory compounds and potency
CA05 4 Weeks 10 mg 5 concentrations tested
release determination.
in quadruplicate
Assay format: Incubations in 48-well plates. ELISA.
Data format: WordTM report with data available in an ExcelTM file.
Test model: Determine effect of compounds on (stimulated)
cytokine release (IL-5) from murine splenocytes. Useful for lead
Up to 3 compounds at
Mediator optimization for pro-inflammatory compounds and potency
CA06 4 Weeks 10 mg 5 concentrations tested
release determination.
in quadruplicate
Assay format: Incubations in 48-well plates. ELISA.
Data format: WordTM report with data available in an ExcelTM file.
Test model: Determine effect of compounds on (stimulated)
cytokine release (IL-6) from murine splenocytes. Useful for lead
Up to 3 compounds at
Mediator optimization for Anti-inflammatory compounds and potency
CA07 4 Weeks 10 mg 5 concentrations tested
release determination.
in quadruplicate
Assay format: Incubations in 48-well plates. ELISA.
Data format: WordTM report with data available in an ExcelTM file.
Test model: Determine effect of compounds on (stimulated)
cytokine release (IL-10) from murine splenocytes. Useful for lead
Up to 3 compounds at
Mediator optimization for Anti-inflammatory compounds and potency
CA08 4 Weeks 10 mg 5 concentrations tested
release determination.
in quadruplicate
Assay format: Incubations in 48-well plates. ELISA.
Data format: WordTM report with data available in an ExcelTM file.
R & D Centre: Plot # 5B, 19th K.M Stone, Hosur Road, Bangalore – 560 100, India.
Tel: 91-080-7832265, 7833092, 7834872, Fax: 91-080-7834369, E-mail: cvc@naturalremedy.com

4. THERAPEUTIC AREA – IMMUNOMODULATION
Turn Minimum
Project
REF# Assay Principle Around Compound Standard Format
Type
Time Required
Test model: Determine effect of compounds on (stimulated)
cytokine release (IL-12) from murine splenocytes. Useful for lead
Up to 3 compounds at
Mediator optimization for Anti-inflammatory compounds and potency
CA09 4 Weeks 10 mg 5 concentrations tested
release determination.
in quadruplicate
Assay format: Incubations in 48-well plates. ELISA.
Data format: WordTM report with data available in an ExcelTM file.
Test model: Determine effect of compounds on (stimulated)
cytokine release (IFN-γ) from murine splenocytes. Useful for lead
Up to 3 compounds at
Mediator optimization for pro-inflammatory compounds and potency
CA10 4 Weeks 10 mg 5 concentrations tested
release determination.
in quadruplicate
Assay format: Incubations in 48-well plates. ELISA.
Data format: WordTM report with data available in an ExcelTM file.
R & D Centre: Plot # 5B, 19th K.M Stone, Hosur Road, Bangalore – 560 100, India.
Tel: 91-080-7832265, 7833092, 7834872, Fax: 91-080-7834369, E-mail: cvc@naturalremedy.com

5. THERAPEUTIC AREA – ALLERGY/PAIN AND INFLAMMATION
Turn Minimum
Project
REF# Assay Principle Around Compound Standard Format
Type
Time Required
Test model: Determine effect of compounds on (stimulated) Nitric
Oxide release from murine macrophages. Useful for lead
Up to 4 compounds at
Mediator optimization for Anti-inflammatory compounds and potency
CA11 4 Weeks 10 mg 5 concentrations tested
release determination.
in triplicate
Assay format: Incubations in 96-well plates. Colorimetric.
Data format: WordTM report with data available in an ExcelTM file.
Test model: Determine effect of compounds on (stimulated)
Prostaglandin release (PGE2) from murine macrophages. Useful
Up to 4 compounds at
Mediator for lead optimization for Anti-inflammatory compounds and
CA12 4 Weeks 10 mg 5 concentrations tested
release potency determination.
in triplicate
Assay format: Incubations in 96-well plates. HTRF.
Data format: WordTM report with data available in an ExcelTM file.
Test model: Determine effect of compounds on (stimulated)
Thromboxane release (TXB2) from Human pro-myelocytic
Up to 4 compounds at
Mediator leukemia cells. Useful for lead optimization for Anti-inflammatory
CA13 4 Weeks 10 mg 5 concentrations tested
release compounds and potency determination.
in triplicate
Assay format: Incubations in 96-well plates. ELISA.
Data format: WordTM report with data available in an ExcelTM file.
Test model: Determine effect of compounds on (stimulated)
Leukotriene release (LTB4) from Human pro-myelocytic leukemia
Up to 4 compounds at
Mediator cells. Useful for lead optimization for Anti-inflammatory
CA14 4 Weeks 10 mg 5 concentrations tested
release compounds and potency determination.
in triplicate
Assay format: Incubations in 96-well plates. HTRF.
Data format: WordTM report with data available in an ExcelTM file.
R & D Centre: Plot # 5B, 19th K.M Stone, Hosur Road, Bangalore – 560 100, India.
Tel: 91-080-7832265, 7833092, 7834872, Fax: 91-080-7834369, E-mail: cvc@naturalremedy.com

6. THERAPEUTIC AREA – ALLERGY/PAIN AND INFLAMMATION
Turn Minimum
Project
REF# Assay Principle Around Compound Standard Format
Type
Time Required
Test model: Determine effect of compounds on (stimulated)
cytokine release (IL-1β) from murine macrophages. Useful for lead
Up to 4 compounds at
Mediator optimization for Anti-inflammatory compounds and potency
CA15 4 Weeks 10 mg 5 concentrations tested
release determination.
in triplicate
Assay format: Incubations in 96-well plates. ELISA.
Data format: WordTM report with data available in an ExcelTM file.
Test model: Determine effect of compounds on (stimulated)
Histamine release from rat basophilic leukemia cells. Useful for
Up to 4 compounds at
Mediator lead optimization for Anti-allergic compounds and potency
CA16 4 Weeks 10 mg 5 concentrations tested
release determination.
in triplicate
Assay format: Incubations in 96-well plates. HTRF.
Data format: WordTM report with data available in an ExcelTM file.
Test model: Determine effect of compounds on (stimulated)
cytokine release (MCP-1) from murine peritoneal macrophages.
Up to 4 compounds at
Mediator Useful for lead optimization for Anti-inflammatory compounds and
CA17 4 Weeks 10 mg 5 concentrations tested
release potency determination.
in triplicate
Assay format: Incubations in 96-well plates. ELISA.
Data format: WordTM report with data available in an ExcelTM file.
R & D Centre: Plot # 5B, 19th K.M Stone, Hosur Road, Bangalore – 560 100, India.
Tel: 91-080-7832265, 7833092, 7834872, Fax: 91-080-7834369, E-mail: cvc@naturalremedy.com

7. THERAPEUTIC AREA – IN VITRO TOXICITY- HEPATOPROTECTION
Turn Minimum
Project
REF# Assay Principle Around Compound Standard Format
Type
Time Required
Test model: Measurement of compound toxicity in hepatocytes
against t-BH using viability (MTT) endpoints. Assess compound
Up to 4 compounds at
In vitro safety before proceeding to clinical development.
CA18 4 Weeks 10 mg 5 concentrations tested
Toxicity Assay format: Incubations in 96-well plates. In vitro toxicity
in triplicate
profiling.
Data format: Word™ report with data available in an Excel™ file.
Test model: Measurement of compound toxicity in hepatocytes
against diazinon (OPC) using viability (MTT) endpoints. Assess
Up to 4 compounds at
In vitro compound safety before proceeding to clinical development.
CA19 4 Weeks 10 mg 5 concentrations tested
Toxicity Assay format: Incubations in 96-well plates. In vitro toxicity
in triplicate
profiling.
Data format: Word™ report with data available in an Excel™ file.
Test model: Measurement of compound toxicity in hepatocytes
against Aldrin (OC) using viability (MTT) endpoints. Assess
Up to 4 compounds at
In vitro compound safety before proceeding to clinical development.
CA19 4 Weeks 10 mg 5 concentrations tested
Toxicity Assay format: Incubations in 96-well plates. In vitro toxicity
in triplicate
profiling.
Data format: Word™ report with data available in an Excel™ file.
Test model: Measurement of compound toxicity in hepatocytes
against Aflatoxin (AFB1) using viability (MTT) endpoints. Assess
Up to 4 compounds at
In vitro compound safety before proceeding to clinical development.
CA20 4 Weeks 10 mg 5 concentrations tested
Toxicity Assay format: Incubations in 96-well plates. In vitro toxicity
in triplicate
profiling.
Data format: Word™ report with data available in an Excel™ file.
R & D Centre: Plot # 5B, 19th K.M Stone, Hosur Road, Bangalore – 560 100, India.
Tel: 91-080-7832265, 7833092, 7834872, Fax: 91-080-7834369, E-mail: cvc@naturalremedy.com

8. THERAPEUTIC AREA – IN VITRO TOXICITY- HEPATOPROTECTION
Turn Minimum
Project
REF# Assay Principle Around Compound Standard Format
Type
Time Required
Test model: Measurement of compound toxicity in hepatocytes
against Galactosamine using viability (MTT) endpoints. Assess
Up to 4 compounds at
In vitro compound safety before proceeding to clinical development.
CA21 4 Weeks 10 mg 5 concentrations tested
Toxicity Assay format: Incubations in 96-well plates. In vitro toxicity
in triplicate
profiling.
Data format: Word™ report with data available in an Excel™ file.
THERAPEUTIC AREA – COSMETOLOGY
Turn Minimum
Project
REF# Assay Principle Around Compound Standard Format
Type
Time Required
Test model: Determine effect of compounds on (un-stimulated)
collagen release from primary human dermal fibroblast. Useful for Up to 4 compounds at
CA22 Anti-wrinkle lead optimization for cosmetology ingredient. 4 Weeks 10 mg 5 concentrations tested
Assay format: Incubations in 96-well plates. Colorimetric. in triplicate
Data format: WordTM report with data available in an ExcelTM file.
Test model: Determine effect of compounds on (un-stimulated)
Melanin release from mouse melanoma cells. Useful for lead Up to 4 compounds at
Skin
CA23 optimization for cosmetology ingredient. 4 Weeks 10 mg 5 concentrations tested
whitening
Assay format: Incubations in 96-well plates. Colorimetric. in triplicate
Data format: WordTM report with data available in an ExcelTM file.
R & D Centre: Plot # 5B, 19th K.M Stone, Hosur Road, Bangalore – 560 100, India.
Tel: 91-080-7832265, 7833092, 7834872, Fax: 91-080-7834369, E-mail: cvc@naturalremedy.com

9. THERAPEUTIC AREA – DIABETES/OBESITY
Turn Minimum
REF# Project Type Assay Principle Around Compound Standard Format
Time Required
Test Model: Measurement of induction of glucose stimulated
Insulin secretion in rat insulinoma cells. This approach is useful for
assessing the effectiveness of compounds under development for Up to 4 compounds at
CA24 GSIS Assay the treatment of type 1 diabetes. 4 Weeks 10 mg 5 concentrations tested
Assay Format: Incubations in 96-well plates. HTRF. in triplicate
Data Format: WordTM report with data available in an ExcelTM
file.
Test model: Determine effect of compounds on (stimulated)
Lipid
Adipocyte differentiation in Mouse embryonic fibroblast . Useful Up to 4 compounds at
Metabolism
CA25 for lead optimization for inhibitors of adipogenesis. 4 Weeks 10 mg 5 concentrations tested
Assays
Assay format: Incubations in 96-well plates. Colorimetric. in triplicate
(Adipogenesis)
Data format: WordTM report with data available in an ExcelTM file.
Test model: Determine effect of compounds on (stimulated)
Lipid
glycerol release in Mouse embryonic fibroblast . Useful for loead Up to 4 compounds at
Metabolism
CA26 identification for inhibitors of adipolysis. 4 Weeks 10 mg 5 concentrations tested
Assays
Assay format: Incubations in 96-well plates. Colorimetric. in triplicate
(Adipolysis)
Data format: WordTM report with data available in an ExcelTM file.
R & D Centre: Plot # 5B, 19th K.M Stone, Hosur Road, Bangalore – 560 100, India.
Tel: 91-080-7832265, 7833092, 7834872, Fax: 91-080-7834369, E-mail: cvc@naturalremedy.com

10. THERAPEUTIC AREA – ONCOLOGY
Turn Minimum
REF# Project Type Assay Principle Around Compound Standard Format
Time Required
Test Model: Determine effect of compounds on cell proliferation
in human breast adenocarcinoma cells. Useful for lead optimization
Anti Up to 4 compounds at
of anti-cancer compound screening.
CA27 proliferative 4 Weeks 10 mg 5 concentrations tested
assay
Assay Format: Incubations in 96-well plates. Colorimetric.
in triplicate
Data Format: WordTM report with data available in an ExcelTM
file.
THERAPEUTIC AREA – GENERAL HEALTH/ HEART DISEASE
Turn Minimum
REF# Project Type Assay Principle Around Compound Standard Format
Time Required
Test Model: Determine the cellular antioxidant potential of
compounds in Human hepatocellular liver carcinoma cells. Useful
cost-effective method to evaluate the antioxidant activity of foods, Up to 4 compounds at
Antioxidant
CA28 dietary supplements, phytochemicals and extracts.. 4 Weeks 10 mg 5 concentrations tested
assay
Assay Format: Incubations in 96-well plates. Colorimetric. in triplicate
Data Format: WordTM report with data available in an ExcelTM
file.
R & D Centre: Plot # 5B, 19th K.M Stone, Hosur Road, Bangalore – 560 100, India.
Tel: 91-080-7832265, 7833092, 7834872, Fax: 91-080-7834369, E-mail: cvc@naturalremedy.com

11. THERAPEUTIC AREA – IN VITRO GENOTOXICITY
Turn Minimum
REF# Project Type Assay Principle Around Compound Standard Format
Time Required
Test Model:. Determine the mutageneic potential of compounds
using 5 starins of S.Typhimurium with and without S9 fractions.
Up to 1 compounds at
Mutagenesis Quick assay to estimate the carcinogenic potential of a compound
CA29 4 Weeks 10 mg 6 concentrations tested
assay Assay Format: Incubations in 384-well plates. Hightroughput.
in duplicate.
Data Format: WordTM report with data available in an ExcelTM
file.
Test model: Determine effect of compounds on the formation of
small membrane bound DNA fragments i.e. micronuclei in the
cytoplasm of interphase cells using Chinese hamster cells. Useful Up to 1 compounds at
Mutagenesis
CA30 for early identification of compounds likely to exhibit genotoxic 15 Weeks 10 mg 3 concentrations tested
assay
liability. in duplicate.
Assay format: Incubations in 90mm petri plates. Manual.
Data format: WordTM report with data available in an ExcelTM file.
Test model: Determine effect of compounds on induction of
structural changes in chromosomes using Chinese hamster cells.
Up to 1 compounds at
Cytogenetic Useful to identify agents that cause structural chromosome
CA31 15 Weeks 10 mg 3 concentrations tested
Assays aberration in cultured mammalian cells.
in duplicate.
Assay format: Incubations in 90mm petri plates. Manual.
Data format: WordTM report with data available in an ExcelTM file.
R & D Centre: Plot # 5B, 19th K.M Stone, Hosur Road, Bangalore – 560 100, India.
Tel: 91-080-7832265, 7833092, 7834872, Fax: 91-080-7834369, E-mail: cvc@naturalremedy.com