Cell Biology Assays

Cell-based assays using either human or animal tissues can be effectively applied to interrogate the biological activity, toxicity or mechanism of action of a test molecule.

Bioassay Target Validation

Target validation ensures that the target is expressed and then confirms if the engagement of the target has any potential therapeutic benefit. It is a critical step in the early phase of drug development.

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Target validation includes the following stages:
  • Discovering a biomolecule of interest
  • Investigating the expression of the molecular target within the tissue of interest
  • Evaluating its potential as a target to understand if it is directly involved in the disease process
  • Designing a bioassay to measure biological activity to verify if target modulation produces the desired therapeutic effect
  • Constructing a high-throughput screen
  • Performing screening to find hits and evaluating the hits
We offer a range of strategies and capabilities for modulating gene expression in vitro. These include but are not limited to the use of antibodies, negative dominant controls, antisense oligonucleotides, ribozymes, and small-interfering RNAs.
Our services to support target validation and discovery:
  • We can offer support and assay design for protein and novel target expression in a range of normal and diseased tissue, including:
    • IHC and novel marker assay development & co-localisation studies
    • Phosphorylated marker assay development
  • High-throughput target validation assay that represents biology
  • Molecular level
    • Screen enzyme inhibitors or activators
  • Cellular Level
    • Verify the involvement of the protein in the disease state (often use gene silencing siRNAs)
    • Understand the protein pathways and interactions

HeLA cells were transfected with the siGLO™ transfector indicator (A), siNegative Control and siWRN according to the manufacturer’s instructions. WRN gene expression was assessed 72 hours post-transfection by Quantigene ™ assay (C) and Western Blot (B) to confirm the siRNA knockdown.

Mechanistic Assays

Understanding the biology of a disease is critical to discovering new therapies for its management. Mechanistic assays offer a valuable tool to get a better understanding of the regulatory processes within a healthy body and to identify the relevant ‘triggers’ that lead to disease development.

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Understanding the biology of a disease is critical to discovering new therapies for its management. Mechanistic assays offer a valuable tool to get a better understanding of the regulatory processes within a healthy body and to identify the relevant ‘triggers’ that lead to disease development.

At Cellomatics, our team of scientific experts can support your mechanistic drug discovery programmes in several ways using 2D or 3D cell culture models:

  1. Identifying the relevant signalling pathway(s)
  2. Identifying/validating the disease biomarker of interest
  3. Understanding the molecular mechanism of action

Pathway activation: Protein Analysis

Singleplex Chemiluminescence imaging of IC-21 cell lysates assayed using the Advanced Q9 Alliance scanner (UVItec Cambridge). IC-21 cells were stimulated with LPS for various time points to analyse the pathway proteins.

Pathway activation: Gene Expression

Cell lines – A431, MCF7 and HeLa were stimulated with EGF, IGF-1 and TNFα respectively. The stimulated cell lysates along with unstimulated lysates were then investigated for multiple signalling pathway activation using Multiplex Quantigene Assay (Luminex).

Toxicity Profiling

A lot of compounds, though efficacious, may fail to meet the acceptable toxicity profile. Toxicity testing is therefore an important facet of drug development. The objective is to ensure that the test substance is safe for further preclinical/clinical development.

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Drugs research and development cost every year over $145 billion in US only. One of the key contributors to these rising costs is unacceptable drug safety profile either detected during Phase 1 Clinical Trial (50-60%) or during Phase IV Post marketing surveillance where some severe side effects were unnoticed during the phase I clinical trial. Of note, it has been described that in 30% of the studied cases the animals showed a toxic effect on a different organ compared to human (https://www.gwern.net/docs/dnb/2000-olson.pdf)

A549 cells were treated with drug compounds at 8 different concentrations. Both Cell Viability and Cytotoxicity of the cells post treatment was assessed using CellTitre-Glo and LDH respectively. Staurosporine (positive compound control) showed a significant reduction in cell viability when compared to untreated or vehicle control (***p<0.001; ±SEM).

HeLa cells were treated with Staurosporine for 4 hours at concentrations of: 10µM, 5µM, 2µM, 1µM, 500nM, 250nM, and 125nM. The Caspase-Glo 3/7® Reagent was added directly to cells in 96-well plates and incubated at room temperature for 30 minutes before recording luminescence (RLU). Each point represents the average of 3 replicates (error bars represent ±SEM).

Cytotoxic profile of a test antibody (1°Ab) in the presence of 2° antibody conjugated with-MMAE toxin. The viability of Kasumi-3 was analysed using Cell-Titer Glo Luminescence assay. A statistically significant decrease in Kasumi-3 cell viability was observed with concentrations above 0.01 nM test antibody in presence of 2°Ab-MMAE when analysed using One-way ANOVA, followed by Dunnett’s post-hoc multiple comparison test (*p<0.02; **p<0.005; ***p<0.0003, <0.0001; ±SEM).
Acute kidney diseaseChronic kidney diseaseNephrotoxicity
Neutrophil gelatinase-associate lipocalin (NGAL)Asymmetric dimethylarginine (ADMA)N-acetyl-β-D-glucosaminidase (NAG)
interleukin-18 (IL18)Neutrophil gelatinase-associate lipocalin (NGAL)Glutathione S-transferases (GST)
Kidney Injury Molecule-1 (KIM1)Kidney Injury Molecule-1 (KIM-1)Gamma-glutamyltransferase (GGT)
Cystatic C (CST3)Liver-type Fatty acid binding protein (L-FABP)Kidney Injury Molecule-1 (KIM1)
Liver-type Fatty acid binding protein (L-FABP)Lactic dehydrogenase (LDH) 
Insulin like growth factor binding protein (IGFBP7)  
Tissue metallopeptidase inhibitor 2 (TIMP-2)  
 
HumanMouseRat
AlbuminClusterinAlbumin
ClusterinCystatin CCalbindin
Collagen IVEpithelial growth factorClusterin
Cystatin CInterferon gamma-induced protein 10Cystatin C
Glutathione S-transferasesKidney Injury Molecule-1Glutathione S-transferases
Interferon gamma-induced protein 10Lipocalin-2Interferon gamma-induced protein 10
Kidney Injury Molecule-1OsteopontinKidney Injury Molecule-1
Lipocalin-2ReninLipocalin-2
Liver-type Fatty acid binding proteinβ-2-MicroglobulinOsteopontin
Macrophage activator proteinTissue metallopeptidase inhibitor 2Tissue metallopeptidase inhibitor 2
Matrix Metalloproteinases (MMP9)Vascular Endothelial Growth FactorVascular Endothelial Growth Factor
Osteoactivin β-2-Microglobulin (β2M)
OsteopontinInterleukin 2,6,8,10 (IL2, IL 6, IL8, IL10)Interleukin 2,6,8,10 (IL2, IL 6, IL8, IL10)
Renin Trefoil factor 3
Trefoil factor 3  
a-1-Microglobulin  
Heat Shock Protein 70 (HSP70)  
Interleukin 2,6,8,10 (IL2, IL 6, IL8, IL10)  
 
HumanRats
Liver-Type Arginase 1 (ARG1)Liver-Type Arginase 1 (ARG1)
Malate dehydrogenase 1 (MDH1)aspartate transaminase 1 (GOT1)
α-glutathione S-transferase (GSTα)α-glutathione S-transferase (GSTα)
Sorbitol Dehydrogenase (SDH)Sorbitol Dehydrogenase (SDH)
5′-Nucleotidase/CD73 (5’-NT)5′-Nucleotidase (5′-NT/CD73).

Request a consultation with Cellomatics Biosciences today

Our experienced team of in vitro laboratory scientists will work with you to understand your cell-based assay development needs and provide a bespoke project plan with a professional, flexible service and a fast turnaround time.

To request a consultation where we can discuss your exact requirements, please contact Cellomatics Biosciences.