Our biological assays can be used for:
- Consumer education Our biological analyses deliver valid preclinical and clinical results that target primary wellness concerns. Function-specific validation benefits consumers and the companies that provide it.
- Product formulation With our reliable suite of biological efficacy tests, companies can build better biologically effective formulations. Our tests deliver valuable guidance in the development of health function-specific formulations—with performance results unique to the actual formulation.
- Claim substantiation Provide in vitro study evidence on function claims
- Quality control (QC) Robust, efficient efficacy tests that address the performance characteristics of natural products provides new QC benchmarks for product manufacturers.
Primary Health Function and Concerns Evaluated
- Biouptake and bioavailability assessment: via human (or canine) cells formed in vitro intestinal model, e.g., Caco-2 model
- Anti-oxidation effect: cellular Nrf2 activation, cellular antioxidant analysis (CAA)
- Oxidative stress reduction: in vivo/in vitro oxidative stress reduction analysis via oxidative stress biomarkers (MDA, GSH/GSSG, isoprostane,s 3-nitrotyrosine, 8-OHdG, SOD, catalase, etc.)
- Anti-inflammatory function: Cellular and enzymatic cytokines (TNF-alpha, interleukins) inhibition, NFκB inhibition, PGE2 inhibition, LOX, COX, and PLA2 inhibition analysis
- Diabetes and weight management: Impact on glucose transport, uptake, and metabolism; Impact on insulin secretion, insulin receptor activity, and carbohydrate digestion
- Immune Boost: via cellular interleukins (ILs) expression
- Anti-allergy property: via cellular Nrf2 assay
- Adaptogenic function via neuronal cellular stress hormone and heat shock protein studies
- Neuroprotective function via neuronal cellular DNA damage and in vivo antioxidants studies
- Cardiovascular and blood pressure via ACE inhibition
- Detoxification and liver function analysis: Phase I and Phase II liver enzymes, liver function biomarkers in serum/plasma
- Energy metabolism: impact on cellular and mitochondrial potential, oxidative phosphorylation, glycolysis, and metabolic gene expression
- Gout Improvement via xanthine oxidase inhibition
- Hair growth and hair loss reduction
- Anti-cancer property: Cancer cell viability, proliferation, and apoptosis analysis
- Anti-aging function: Cellular SIRT1 enhancement; Cellular telomerase activity
- Mitochondria health: mitochondrial ROS level
- Neurite Outgrowth Assay: assesses the neurotic outgrowth using a widely used neuronal model based on neuron growth factor (NGF)-stimulated mammalian cells
- Cytotoxicity: cellular viability assay (CVA)
Study Models applied:
- Human and mamalian cell lines: HepG2, PC3, beta-cells, (L929, Vero, IMR-90, MRC-5, CHO, HL-60, etc.)
- Primary cells: hepatocytes, primary fibroblast or keratinocyte cultures
- Caco-2: human intestine model
- Isolated human blood cells: PBMC, lymphocytes, etc.
- 3D skin model: full thickness epidermis
- Microtissues: liver microtissue, primary pancreatic islets
Overiew of Each Biossay Investigation
1. BioUptake and BioAvailability Assessment: via human (or canine) intestinal cells formed in vitro intestine model
Many substance are absorbed by passive transport through the intestine membrane into blood. Such trans-cellular permeability properties can significantly influence the oral absorption of a food/nutritional product or medicine candidate. Intestinal permeability is a critical characteristic that determines the rate and extent of human absorption and ultimately affects the bioavailability of a nutritional product or drug candidate.
1.1 Caco-2 assay: biouptake/availability assessment via human intestinal membrane Caco-2 cells are human intestinal cells that are widely used to measure biouptake and absorption capability of a food and nutritional product. When cultured under specific conditions they become differentiated and polarised such that morphologically and functionally resemble the epithelial cells of human small intestine, and the monolayers of caco-2 cells resemble the membrane of human intestines.
The Caco-2 permeability assay is considered to be the golden standard method in nutraceutical and pharmaceutical industries for in vitro prediction of in vivo human intestinal bioabsorption and biouptake of a food/nutritional product or medicine candidate. In drug discovery field, Caco-2 permeability assay is also used to predict drug-drug interactions (DDIs) of orally administered drugs. The FDA recommends that drug-drug interactions should be performed during drug development (FDA Guidance for Pharmaceutical Industry). The Caco-2 Permeability Assay utilizes a Caco-2 cell layer grown on a supportive membrane surface that separates two compartments. Typically, the substance to be studied is added to the apical side of a confluent monolayer of Caco-2 cells and permeability is measured by monitoring the appearance of the test compound on the opposite side (basolateral side) of the monolayer.
1.2 MDCK assay: biouptake/availability assessment via canine epithelial cells Madin Darby canine kidney (MDCK) cells are an epithelial cell line of canine kidney origin. Cultured on semiporous filters, the MDCK cells form confluent monolayers that model the intestinal epithelial barrier for permeability, transporter and drug-interaction assays. MDCK assay is a mature cellular model in pharmaceutical industry that provides qualitative prediction of absorption, determines mechanism(s) of permeability and formulation effects on drug permeability. We provide MDCK assay as an alternative of caco-2 permeability assay, well as a key model for pet food industry for its biouptake and bioavailability assessment.
2. Antioxidant effect Cellular antioxidant investigation defines a test material’s intracellular antioxidant capacity in a cellular context. It is also a preclinical measure of bioavailability of a test article that describes the efficiency of absorption by human cells. Two biological investigations are offered in this function area:
2.1 Cellular Nrf2 activation Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that binds to antioxidant response elements (ARE) to regulate the expression of antioxidant enzymes that protect against oxidative damage triggered by injury and inflammation. Activation of the Nrf2 pathway has been found to prevent and treat a large number of chronic inflammatory diseases. A number of naturing occurring phytonutrients such as resveratrol, sulforaphane, and curcumin have been reported to activate Nrf2, with more phytonutrient Nrf2 activator discoveries being the focus of investigation. Nrf2 has been investigated as biomarker for regulating in vivo anti-oxidation and anti-inflammation response. In our cellular Nrf2/ARE investigation, we monitor the impact of a test material on Nrf2 activation to assess the material’s anti-oxidation and anti-inflammatory capacity using human cells.
2.2 Cellular Antioxidant Analysis (CAA) Cellular Antioxidant Analysis (CAA) assay has been used in food and nutritional industry as a mean to evaluate the antioxidant protection capacity of a material again oxygen radical challenge. In CAA assay, a fluorescent probe is placed inside of representative human cells, whose loss of fluorescence is an indication of the damage extent from oxygen radical. A material to be tested is incubated with the cells to allow its natural absorption into cells. Then, an oxygen radical inducer is introduced into cellular environment, which triggers the release of oxygen radicals. Without antioxidant material present inside of the cells, oxygen radicals permeate through cell membrane and damage the cells and the marker probe. Such process deters when antioxidant material is present inside of the cells. The cellular antioxidant effect of the test material is then measured by assessing the preservation effect of the marker probe in the presence of the test material absorbed inside of cells.
3. Oxidative stress reduction: in vivo and in vitro oxidative stress reduction analysis
We offer oxidation stress analysis in conjunction with a clinical study, preclinical (animal) study, or cellular investigation. Blood (plasma or serum), urine, tissue or cell lysate samples are taken from participating subjects/cells and oxidative stress analyses are performed to assess the impact of a material on the oxidative stress condition of the participating subjects/cells.
For a general assessment of oxidative stress level in a biological system, two key areas of the investigation are recommended: in vivo antioxidant; and level of stress damage biomarkers. In vivo antioxidant indicates the defense level in a biological system, while damage biomarkers assess the level of the damage a biological system experiences from oxidative stress. Through assessing these two complementary areas we can provide an overall evaluation of oxidative stress condition in a biological system. We offer following complete, full panel of oxidative stress biomarker analysis via both LC-MS/MS and spectrometric/ELISA methods for most of the biomarkers:
3-1. in vivo antioxidants: Super Oxide Dismutase, Catalase, Glutathione (GSH); Toal Glutathinone (GSH+GSSG); (GSH/GSSG), Glutathione Peroxidase (GPx), Glutathione S-transferase (GST), Glutathione Reductase, In vivo ORAC
3-2. Damage Markers from Oxidative Stress: Damages of a biological system induced by oxidative stress mainly reflect in three areas: lipid peroxidation, protein damage, and DNA damage. Major biomarkers in these areas are:
- Lipid peroxidation: TBARS (Thiobarbituric Acid Reactive Substances), Isoprostanes (F2-isoprostanes, F2-isoPs), Malondialdehyde (MDA), 4-Hydroxynonenal (4-HNE), Lipid hydroperoxide (LOP)
- Protein damage: 3-Nitrotyrosine, Advanced Glycation End Products (AGE), Protein Carbonyl (PCO)
- DNA/RNA oxidation: 8-Hydroxydeoxyguanosine (8-OHdG), 8-Hydroxyguanosine (8-OHG)
4. Anti-inflammatory function: via cytokines (TNF-alpha, interleukins), NFkB inhibition, PGE2, LOX and COX inhibition analysis
Inflammation plays a key role in the progression of many diseases, including rheumatoid arthritis, cancer, atherosclerosis and other cardiovascular diseases, and inflammation regulation and inhibition have been found as key therapeutic approaches to these disease conditions. The mechanism of inflammation formation is complex, involving various cellular signaling processes and paths. A number of inflammation markers have been discovered as targets for different anti-inflammatory approaches. Each marker has its own mechanism in inducing or regulating inflammation, with its function often cross relating to functions of other markers. Therefore the impact of a material on inflammation is better understood via multiple marker evaluation. Listed below are key biomarkers of inflammation, and mammalian cells based investigation we offer to assess potential anti-inflammatory properties of a nutraceutical material through its impact on these key markers.
4.1. Cellular cytokines (TNF-alpha) inhibition: Tumor necrosis factor (TNF)-alpha is a proinflammatory cytokine (small protein impacting cell signaling) that triggers downstream cellular feedback loops governing inflammation. TNF-alpha has been identified as an inflammation trigger and precursor. In many anti-inflammation investigation, TNF-alpha inhibitors are sought to function as anti-inflammatory agents that has potential treatment effect for rheumatoid arthritis (RA) and other inflammatory diseases. At present, clinically proven TNF-alpha inhibitors are protein-based drugs such as infliximab and etanercept for inflammation diseases treatment. However, these protein-based drugs have potential devastating side effects such as heart failure and neurological changes. Natural plants have therefore become of investigative interests to explore their role as less toxic TNF-alpha inhibitors for inflammation treatment, with natural flavonoids such as quercetin and catechin being shown as TNF-alpha inhibitors without significant known side effects. In this investigation, we focus on TNF-alpha inhibition effect of a nutraceutical material to evaluate its its dietary functional function in anti-inflammation area.
4.2. Cellular cytokines (Interleukins) inhibition: Similar to TNF-alpha, interleukins are inflammatory cytokines that are also of keen interests to be investigated as biomarkers for inflammation. Cellular inhibition of interleukins can be investigated through similar cellular investigation discussed above. Inhibition of five main interleukins, IL-2, IL-4, IL-6, IL-8, and IL-10 as well-documented pro-inflammatory cytokine markers can be investigated via these analyses.
4.3. Cellular NF-κB inhibition: NF-κB (Nuclear Factor kappa B), a protein complex that is involved in cellular responses to stimuli such as stress and free radicals, ultraviolet irradiation, oxidized LDL, and bacterial or viral antigens. It plays a key role in regulating the immune response to infection. Suppression of NF-κB limits the production of proinflammatory gene expression and reduces the level of inflammation, therefore NF-κB has been studied as a biomarker for inflammation, and inhibition of NF-κB has been used as an indicator for anti-inflammatory potential. A classic example of anti-inflammatory material via NF-κB inhibition are natural compounds such as curcumin and green tea polyphenols that have been demonstrated to inhibit inflammation via NF-κB inhibition. In this assay investigation, we focus on NF-κB inhibition evaluation of a nutraceutical material for dietary functional effect in anti-inflammation area.
4.4. Cellular PGE2 Inhibition: Prostaglandin E2 (PGE2) is a primary product of arachidonic acid metabolism controlled by cyclooxygenase enzymes. It plays a critical role in increasing vascular permeability, fever generation, and tumor growth. Drugs used to inhibit PGE2 synthesis have shown to control inflammation, pain and fever. Under this investigation, we investigate the impact of a material on PGE2 production level in mammalian cells.
4.5. Enzymatic cycloooxygenase (COX) Inhibition: Cyclooxygenases inhibitors are among the important targets for treatment of inflammation related diseases. COX has two well-known isoforms, COX-1 and COX-2, which are similar in their amino-acid sequences and identity. COX-2 predominates at sites of inflammation, and COX-1 is constitutively expressed in most tissues including gastrointestinal tract. It is reported that selective COX-2 inhibitors can target inflammation and pain with reduced risk of chronic ulceration and acute injury, where COX-1 inhibitors typically possess anti-inflammation effect but with gastric side-effects (e.g., hemorrhagic side effect). In our investigation, we concentrate on assessing both COX-1 and COX-2 inhibition capability of a material via enzymatic assays.
4.6. Lipooxygenase (LOX) Inhibition: Lipoxygenases (LOXs) are a family of nonheme iron-containing dioxygenases enzymes distributed in animals, plants, and fungi. These enzymes catalyze distinct cellular reactions and produce fatty acid hydroperoxides throughout the reaction processes. These fatty acid hydroperoxides products have been identified as mediators of a series of inflammatory diseases including rheumatoid arthritis, inflammatory bowel disease, atherosclerosis and certain types of cancer. Therefore LOX inhibitors have been discovered as beneficial to inflammation treatment. Another significance in LOX inhibition is that LOX 5 inhibitors have been identified to potentially counteract the gastric damage associated with the COX-1 inhibitors. This makes LOX 5 inhibitors potential side effect removers for many COX-1 inhibitor anti-inflammatory drugs including aspirin. Through this investigation we determine the inhibition potential of a material on LOX-5 or LOX-15 activity via enzymatic or cellular analysis.
4.6. Secretory Phospholipases A2 (sPLA2) Inhibition: Secretory Phospholipases A2 (sPLA2) are a subfamily of Phospholipases A2 enzymes that catalyze the hydrolysis of phospholipids yielding precursors of pro-inflammatory lipid mediators including bioactive eicosanoids and platelet-activating factor (PAF). sPLA2 inhibitors hold an established role in inflammation treatment, since inhibition of sPLA2 in theory would prevent the formation of inflammatory eicosanoids prior to the cyclooxygenase (COX) reaction. Therefore, theoretically, sPLA2 inhibitors eliminate the need for COX inhibitors in anti-inflammatory therapeutics. Through sPLA2 inhibition investigation we determine the inhibition potential of a material on sPLA2 activity via enzymatic analysis.
5. Diabetes and weight management Glucose regulation is a functional process that under degenerate conditions may result in various health issues including obesity, diabetes, and cancer. Metabolically, the insulin receptor, a transmembrane receptor that is activated by insulin and insulin-like growth factors, plays a key role in the glucose regulation. Under decreased insulin secretion or insulin receptor activity condition, the cells are unable to take up glucose, resulting hyperglycemia (an increase in circulating glucose), leading to diabetes mellitus type 2 (commonly called Type 2 diabetes). Therefore, materials that are able to inhibit glucose transport into blood stream or enhance cellular glucose uptake, enhance insulin secretion and insulin receptor activity are helpful to improve diabetic condition and weight management.
On a separate direction, a proven successful approach in diabetic condition improvement and weight management has been the disruption of nutrient digestion. Investigation has been driven towards nutraceuticals that inhibit the breakdown of complex carbohydrates and fats within the gut. Materials inhibit activity of α-amylase, an enzyme that catalyses the hydrolysis of starch into sugars, deter carbohydrate digestion therefore have been used in humans with promising results relating to diabetic condition improvement and weight loss.
Therefore, we carry out preclinical evaluation on cells and pancreatic islets in following three areas to understand the potential diabetes and weight management function of a nutraceutical material:
5.1. Glucose uptake: Glucose uptake from intestine to blood stream via caco-2 human intestine cells; Glucose uptake into cells and glucose metabolism using liver cells
5.2 Insulin secretion and insulin receptor activity: Bioassays via cells and primary pancreatic islets; Insulin secretion assay in mammalian cells or primary pancreatic islets; Cellular insulin receptor assay in mammalian cells using Akt phosphorylation as the biomarker.
5.3 Carbohydrate digestion inhibition: Enzymatic alpha-amylase inhibition assay, Enzymatic alpha-glucosidase inhibition assay
6. Immune Boost: cellular interleukins (ILs), TNF-α inhibition assay
The innate immune system consists of a wide variety of biological processes that activate quickly to defend against invading pathogens. Cytokines are regulatory proteins produced by the immune system and function as cellular mediators of immune responses and inflammatory reactions. In autoimmune diseases, an abnormal production of proinflammatory cytokines such as tumor necrosis alpha (TNF-alpha) and interleukins may lead to an imbalance. The combination of cytokine inhibitors had been found a potential therapeutic approach in the treatment of immune-mediated diseases including rheumatoid arthritis. Under this investigation, cellular inhibition of interleukin-1 beta (IL-1β) is utilized as a model for immune function assessment.
Similar to IL-1β, TNF-α and other interleukins (e.g., IL-6) are inflammatory cytokines that are also of keen interests to be investigated as biomarkers for immune-mediated diseases.
7. Anti-allergy function: via cellular Nrf2 enhancement assay
Allergy includes a number of conditions caused by hypersensitivity of the immune system responding to the environment. Well-known allergic conditions include asthma, hay fever, etc., that have been found triggered by environment-induced chronic inflammation in airway or skin. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that binds to antioxidant response elements (ARE) to regulate the expression of antioxidant enzymes that protect against oxidative damage triggered by injury and inflammation. Previous in vivo and in vitro studies have also shown that disruption of Nrf2 expression stimulates airway inflammation and hyper-responsiveness in a mouse model of allergic asthma, indicating the important protective role that Nrf2 plays in allergic and asthmatic responses of a biological system. In this assay, we study the impact of a test material on cellular Nrf2 expression to assess its potential anti-allergic function.
8. Detoxification and liver function analysis Phase I and Phase II liver enzymes, liver function biomarkers in serum/plasma
Liver detoxifies via two major detoxification pathways: Phase I and Phase II. Phase I detoxification converts a toxic chemical into a less harmful chemical via oxidation reduction and hydrolysis. Phase I detoxification is catalysed by enzymes commonly referred to as the cytochrome P450 enzyme group (or Mixed Function Oxidase enzymes MFO). The induction of P450 enzymes have been used as an indicator of enhanced liver Phase I detoxification capability.
Phase II is the second step in processing toxic compounds for elimination. In this phase, the liver adds small chemical pieces onto the toxins via conjugation process to prepare toxins for release into bile or urine. Phase II detoxification requires a number of key enzymes (Phase II enzymes) including glutathione-S-transferases (GSTs), UDP-glucuronyl transferase (UGT), etc.. We provide a panel of assays investigating the detoxification/liver function of a material via its capability in activating Phase I and Phase II liver enzymes in human liver tissues
Phase I enzymes induction in human liver microtissues:
(a) Activities of different cytochrome P450 (CYP) isoforms will be determined by incubating liver microtissues with according substrates and determining substrate consumption levels via LC/MS based P450 assays. Seven major cytochrome P450 (CYP) isoforms will be investigated: CYP1A, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. Other isoforms are available if requested.
(b) The expression levels of CYP will be assessed by qRT-PCR analysis or LC/MS-MS technique.
Phase II enzymes: Expression levels of major Phase II enzymes including glutathione S-transferases (GST), UDP-glucuronyl transferase (UGT), hemeoxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO1), Glutamate-cysteine ligase (GCL), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase(GR), and quinone reductase (QR).
9. Energy metabolism We investigate the health effect of a material on energy metabolism via mammalian cell mode in following four aspects of energy metabolism:
9.1. Cellular reduction potential and Mitochondrial membrane potential Metabolically active cells create a reduction potential that can be quantified and used to indicate cell metabolic activity and cell viability. When measured in a population of cells, cellular reduction potential is proportional to the number of living cells. In the presence of exogenous compounds, decreases in cellular reduction potential can indicate decreased viability. In the meantime, ATP (adenosine triphosphate) generation occurs primarily in mitochondria, and a decrease in mitochondrial membrane potential will lead to a reduced ATP production, and eventually activation of a series of metabolic activities. By assessing cellular and mitochondrial reduction potential in the presence and absence of the test material, we would be able to assess its effects on cellular metabolic activity.
9.2. Oxidative phosphorylation: Cellular oxidative consumption rate assay Energy metabolism is the process by which nutrients are converted to ATP. There are two significant pathways of cellular ATP production: oxidative phosphorylation and glycolysis. Oxidative phosphorylation generates the majority of ATP in an organism and is the result of electron transfer from substrates in the mitochondria. This can be investigated via cellular oxidative consumption rate assay.
9.3. Glycolysis: Glycolysis is another significant pathways of cellular ATP production where glucose is converted into pyruvate and energy is released in the process to form ATP and other high-energy molecules. Degree of glycolysis can be measured by rate of hexokinase formation (Cellular hexokinase assay) and extracellular acidification (Cellular Eextracellular Acidification Assay).
9.4. Metabolic enzymatic activity and gene expression: Substances that substantially increase energy metabolism may have potent effects on gene expression. For example, caffeine activates AMPK (5’adenosine monophosphate-activated protein kinase), which serves as a “fuel gauge” in cells and is activated under conditions that signify cellular stress. Caffeine also significantly increases expression of an essential precursor to mitochondrial biosynthesis, peroxisome proliferator-activated receptor coactivator 1 alpha (PGC-1α). Along the same approach, we apply Cellular AMPK assay and Cellular metabolic gene expression assay to assess whether a test material has significant effects on proteins associated with energy metabolism.
10. Anti-cancer property Cancer cell viability, proliferation, and apoptosis analysis
Cancer is mainly characterized by uncontrolled cell growth and metastasis of tumor cells. In normal human body, cell maintains homeostasis by regulating many attributes of cellular behavior such as proliferation and apoptosis. In cancer cells, the balance between proliferation and death is broken, resulting in the generation and unchecked growth of tumor. Metastasis is very common in the late stage of cancer, which is also the primary cause of mortality in most cancer patients. Cancer metastasis is a process where tumor cells leave the primary tumor and travels through lymph and blood circulatory system and establishes secondary tumor in other body organs. The growth and metastasis of cancer cells can be delayed by many natural extracts. In our investigation, we look into the impact of a material on cancer cell viability, proliferation, and apoptosis to evaluate its potential in cancer treatment area:
10.1 Cancer Cell Viability Assay: Anti-cancer materials inhibit cancer cell growth and kill them by mediating the proliferation and apoptosis. The effect can be detected by measuring the number of living cells after treatment with a rest material. Metabolic activity is a characteristic of living cells. Based on this premise, the number of living cells can be detected by metabolic activity assay. However, this assay only reflects the total number of living cells after treatment, but can’t distinguish which cellular process, proliferation or apoptosis, is changed. Therefore, other specific assays , proliferation and apoptosis analysis, are needed for confirmation of potential anti-cancer properties of a test material.
10. 2. Cancer cell anti-proliferation assay: Cell proliferation is the measurement of the number of cells that are dividing in a culture. The process of cell division, called cell cycle, has four major phases including G1, S, G2 and M phase. DNA replication in S phase is a typical characteristic of cell division. Therefore, the measurement of DNA synthesis is a good marker for proliferation and is used in our assay for caner cell proliferation analysis.
10.3. Cancer cell apoptosis assay: Apoptosis is a carefully regulated process of cell death that occurs as a normal part of development. Inappropriately regulated apoptosis is implicated in many diseases, such as cancer. Promoting apoptosis of cancer cells is a classic strategy for cancer drug discovery. Apoptosis is distinguished from necrosis by morphological and biochemical changes. Based on these characteristics, many different approaches can be used to study apoptosis.
10.4. Cancer cell invasion assay: Invasion of cancer cells into surrounding tissue and the vasculature is an initial step in tumor metastasis. Transwell migration assays have been widely used for studying the motility of different types of cells including metastatic cancer cells. The assay employs a permeable layer of support, usually a tissue-culture-treated microporous membrane, which is positioned between two compartments that mimic two different sets of micro-environments for cell survival/growth. Cells on one side of the membrane, when sensing chemoattractants placed on the other side of the compartment that diffuses through the membrane, can migrate through the pores in the membrane towards the source of the chemoattractants. Cells that migrate across the membrane can be quantified by fixing and counting. It is an effective method to assess the inhibition activity of samples against the cancer migration.
11. Anti-aging function: Cellular SIRT1 enhancement; Cellular telomerase activity
SIRT1 is a protein that is believed to play important roles in longevity and age-related diseases. Previous studies have shown that when cells age, SIRT1 expression decreases, while induction and activation of SIRT1 has been associated with extended life span. These studies have triggered the search for SIRT1 activators that may be used as dietary supplements to promote health and longevity.
Here we investigate potential anti-aging effect of a material via cellular anti-aging analysis using SIRT1 as a biomarker. In this investigation, we determine the ability of a material in stimulating SIRT1 protein expression or activity in human cells, which translates to its anti-aging potential. Human cells will first be treated with and without a test material. Then, SIRT1 production of cells are monitored as they age, and anti-aging properties assessed by comparing the SIRT1 production.
Another area for anti-aging investigation is through telomere and telomerase activity interrogation. Significant amount of research has related telomere length with cellular aging. Telomeres become shorter every time a cell divides, and when they are lost cells can not reproduce further. Telomerase, also called telomere terminal transferase, is an enzyme that carries its own RNA that is use as a template when it elongates telomere. Enhanced telomerase activity have been related to telomere lengthening and aging process deterring. As a result, materials that can enhance telomerase activity have been targets for potential function in anti-aging.
12. Mitochondria health: mitochondrial ROS assay
Mitochondrial ROS Assay assesses the impact of a test material on mitochondria health using the level of mitochondrial reactive oxygen species (mtROS) as a biomarker. High level of ROS, consisting of peroxides, superoxides, hydroxyl radicals, and singlet oxygens, can cause oxidative stress. The vast majority of ROS in cells are generated directly or indirectly by mitochondrial metabolism. mtROS can cause damage to nucleic acids, proteins and lipids. Significantly, mtROS is elevated in a number of adverse conditions, such as cardiovascular disease, cancer, and autoimmune disorders. Therefore, the level of mtROS is a director indicator of mitochondria health, and materials that can decrease mitochondrial ROS levels have potential in improving mitochondria health.
13. Cytotoxicity: cellular viability assay (CVA)
Cytotoxicity of a material is important to determine to provide insight of its biocompatibility with a biological system. The Cell Viability Analysis (CVA) method evaluates the cytotoxicity of a material basing on proliferation investigation that determines the effect of this material on cell growth and cell viability.
In CVA analysis, human cells are treated with or without a series of concentrations of test material. The degree of cell viability is then assessed through monitoring the amount of cellular adenosine triphosphate (ATP), which functions as a biomarker for metabolically active cells, i.e., viability of the cells. The maximum concentration of the material used in the cell treatment that maintains or promotes cell viability is reported.