Assays

Cardio Assays

Vala Assay: #CT1

Cell Type: Human cardiomyocytes derived from hiPSCs

Main Goal of Assay: Determine the effects of test compounds on calcium transient kinetics in human cardiomyocytes derived from induced pluripotent stem cells. Transient increases in cardiomyocyte intracellular calcium concentration translate the cardiac action potential into contractile force. Disruptions in calcium transients can disrupt contractile activity. This assay measures the fluorescence of calcium indicator Fluo-4 at 30 Hz for 10 seconds to capture calcium transient activity on a cell-by-cell basis. Vala’s CyteSeer® image analysis program then reports a range of calcium transient kinetic parameters (transient duration at multiple decay points, rise and decay times, upstroke and downstroke velocities, etc.) to provide a comprehensive picture of how each compound affects the cardiac calcium transient.

CyteSeer® Data Readout

  • Fluorescence channel #1 (DAPI): Hoechst (cell number)
  • Fluorescence channel #2 (green channel): Fluo-4 (calcium transient kinetic measurements)

Vala Assay: #CT2

Cell Type: Human cardiomyocytes derived from hiPSCs

Main Goal of Assay: Determine the effects of test compounds on action potential kinetics in human cardiomyocytes derived from induced pluripotent stem cells. Transient electrical depolarizations, or action potentials, direct cardiomyocyte contractile activity, and disruptions in action potentials can disrupt contraction. This assay measures the fluorescence of membrane potential indicator FluoVolt at 30 Hz for 10 seconds to capture action potentials on a cell-by-cell basis. Vala’s CyteSeer® image analysis program then reports a range of action potential kinetic parameters (duration at multiple decay points, rise and decay times, upstroke and downstroke velocities, etc.) to provide a comprehensive picture of how each compound affects the cardiac action potential.

CyteSeer® Data Readout

  • Fluorescence channel #1 (DAPI): Hoechst (cell number)
  • Fluorescence channel #2 (green channel): FluoVolt (action potential kinetic measurements)

Vala Assay: #CT3

Cell Type: Human cardiomyocytes derived from hiPSCs

Main Goal of Assay: Determine the effects of test compounds on calcium transient kinetics and contractility in human cardiomyocytes derived from induced pluripotent stem cells. Transient increases in cardiomyocyte intracellular calcium concentration translate the cardiac action potential into contractile force. Disruptions in calcium transients can disrupt contractile activity. This assay measures the fluorescence of calcium indicator Fluo-4 at 30 Hz for 10 seconds to capture calcium transient activity on a cell-by-cell basis. Vala’s CyteSeer® image analysis program then reports a range of calcium transient kinetic parameters (transient duration at multiple decay points, rise and decay times, upstroke and downstroke velocities, etc.) to provide a comprehensive picture of how each compound affects the cardiac calcium transient. CyteSeer® also detects the motion of Fluo-4-labeled features within each cardiomyocyte to report kinetic measurements on the contraction that results from each calcium transient.

CyteSeer® Data Readout

  • Fluorescence channel #1 (DAPI): Hoechst (cell number)
  • Fluorescence channel #2 (green channel): Fluo-4 (calcium transient and contractile activity kinetic measurements)

Vala Assay: #CT4

Cell Type: Human cardiomyocytes derived from hiPSCs

Main Goal of Assay: Determine the effects of test compounds on action potential kinetics and contractility in human cardiomyocytes derived from induced pluripotent stem cells. Transient electrical depolarizations, or action potentials, direct cardiomyocyte contractile activity, and disruptions in action potentials can disrupt contraction. This assay measures the fluorescence of membrane potential indicator FluoVolt at 30 Hz for 10 seconds to capture action potentials on a cell-by-cell basis. Vala’s CyteSeer® image analysis program then reports a range of action potential kinetic parameters (duration at multiple decay points, rise and decay times, upstroke and downstroke velocities, etc.) to provide a comprehensive picture of how each compound affects the cardiac action potential. CyteSeer® also detects the motion of FluoVolt-labeled features within each cardiomyocyte to report kinetic measurements on the contraction that results from each action potential.

CyteSeer® Data Readout

  • Fluorescence channel #1 (DAPI): Hoechst (cell number)
  • Fluorescence channel #2 (green channel): FluoVolt (action potential and contractile activity kinetic measurements)

Vala Assay: #CT5

Cell Type: Cardiomyocytes isolated from adult canines

Main Goal of Assay: Determine the effects of test compounds on calcium transient kinetics and contractility in cardiomyocytes from adult dogs, which have similar heart rates and cardiac electrophysiology to humans. Transient increases in cardiomyocyte intracellular calcium concentration translate the cardiac action potential into contractile force. Disruptions in calcium transients can disrupt contractile activity. This assay measures the fluorescence of calcium indicator Fluo-4 at 30 Hz for 10 seconds to capture calcium transient activity on a cell-by-cell basis for electrically paced cardiomyocytes. Vala’s CyteSeer® image analysis program then reports a range of calcium transient kinetic parameters (transient duration at multiple decay points, rise and decay times, upstroke and downstroke velocities, etc.) to provide a comprehensive picture of how each compound affects the cardiac calcium transient. CyteSeer® also detects changes in cardiomyocyte length to report kinetic measurements on the contraction that results from each calcium transient.

CyteSeer® Data Readout

  • Fluorescence channel #1 (DAPI): Hoechst (cell number)
  • Fluorescence channel #2 (green channel): Fluo-4 (calcium transient and contractile activity kinetic measurements)

Vala Assay: #CT6

Cell Type: Cardiomyocytes isolated from adult canines

Main Goal of Assay: Determine the effects of test compounds on action potential kinetics and contractility in cardiomyocytes from adult dogs, which have similar heart rates and cardiac electrophysiology to humans. Transient electrical depolarizations, or action potentials, direct cardiomyocyte contractile activity, and disruptions in action potentials can disrupt contraction. This assay measures the fluorescence of membrane potential indicator FluoVolt at 30 Hz for 10 seconds to capture action potentials on a cell-by-cell basis for electrically paced cardiomyocytes. Vala’s CyteSeer® image analysis program then reports a range of action potential kinetic parameters (duration at multiple decay points, rise and decay times, upstroke and downstroke velocities, etc.) to provide a comprehensive picture of how each compound affects the cardiac action potential. CyteSeer® also detects changes in cardiomyocyte length to report kinetic measurements on the contraction that results from each action potential.

CyteSeer® Data Readout

  • Fluorescence channel #1 (DAPI): Hoechst (cell number)
  • Fluorescence channel #2 (green channel): FluoVolt (action potential kinetic measurements)

Vala Assay: #CT7

Cell Type: Cardiomyocytes isolated from adult canines

Main Goal of Assay: Determine the effects of test compounds on sarcomere contraction in cardiomyocytes from adult dogs, which have similar heart rates and cardiac electrophysiology to humans. In cardiomyocytes, contractile proteins are organized into repeating structures called sarcomeres that coordinate force production across the cell. This assay detects changes in sarcomere spacing in brightfield images captured at 30 Hz for 10 seconds on a cell-by-cell basis. Vala’s CyteSeer® image analysis program then reports a range of sarcomere shortening kinetic parameters (duration at multiple decay points, rise and decay times, upstroke and downstroke velocities, etc.) to provide a comprehensive picture of how each compound affects intracellular cardiomyocyte contraction.

CyteSeer® Data Readout

  • Brightfield: changes in sarcomere spacing over time

Angiogenesis Assays

Vala Assay: #TUBHMC1

Cell Type: HMEC-1 cells derived from human microvascular endothelial cells, which line the capillaries

Main Goal of Assay: Determine the ability of test compounds to enhance capillary network formation in growth-limiting conditions. Capillary growth supports angiogenesis, which is critical for development, maturation, and reproductive function. Dysregulation of angiogenesis can lead to birth defects, macular degeneration, impaired wound healing, and tumor growth. This assay measures compound effects on capillary network formation in basal medium, which lacks growth factors that support capillary growth. Tube formation agonists will increase capillary growth in these conditions.

CyteSeer® Data Readout

  • Fluorescence channel #1 DAPI: cell number, ploidy
  • Fluorescence channel #2 (green channel): cell-permeable cytoplasm dye (length, area, and continuity of objects in the capillary network)

Vala Assay: #TUBHMC2

Cell Type: HMEC-1 cells derived from human microvascular endothelial cells, which line the capillaries

Main Goal of Assay: Determine the ability of test compounds to inhibit capillary network formation in growth-supporting conditions. Capillary growth supports angiogenesis, which is critical for development, maturation, and reproductive function. Dysregulation of angiogenesis can lead to birth defects, macular degeneration, impaired wound healing, and tumor growth. This assay measures compound effects on capillary network formation in complete medium, which contains growth factors that support capillary endothelial cell proliferation. Tube formation antagonists will decrease capillary growth in these conditions.

CyteSeer® Data Readout

  • Fluorescence channel #1 DAPI: cell number, ploidy
  • Fluorescence channel #2 (green channel): cell-permeable cytoplasm dye (length, area, and continuity of objects in the capillary network)

Vala Assay: #TUBHUV1

Cell Type: Primary human umbilical vein-derived endothelial cells (HUVEC-1)

Main Goal of Assay: Determine the ability of test compounds to enhance vein formation in growth-limiting conditions. Vein growth supports angiogenesis, which is critical for development, maturation, and reproductive function. Dysregulation of angiogenesis can lead to birth defects, macular degeneration, impaired wound healing, and tumor growth. This assay measures compound effects on vein formation in basal medium, which lacks growth factors that support blood vessel growth. Tube formation agonists will increase vessel growth in these conditions.

CyteSeer® Data Readout

  • Fluorescence channel #1 DAPI: cell number, ploidy
  • Fluorescence channel #2 (green channel): cell-permeable cytoplasm dye (length, area, and continuity of objects in the capillary network)

Vala Assay: #TUBHUV2

Cell Type: Primary human umbilical vein-derived endothelial cells (HUVEC-1)

Main Goal of Assay: Determine the ability of test compounds to inhibit vein formation in growth-supporting conditions. Vein growth supports angiogenesis, which is critical for development, maturation, and reproductive function. Dysregulation of angiogenesis can lead to birth defects, macular degeneration, impaired wound healing, and tumor growth. This assay measures compound effects on vein formation in complete medium, which contains growth factors that support vein endothelial cell proliferation. Tube formation antagonists will decrease vessel growth in these conditions.

CyteSeer® Data Readout

  • Fluorescence channel #1 DAPI: cell number, ploidy
  • Fluorescence channel #2 (green channel): cell-permeable cytoplasm dye (length, area, and continuity of objects in the capillary network)

Vala Assay: #TUBHMV1

Cell Type: Primary human microvascular-derived endothelial cells (HMVEC-1)

Main Goal of Assay: Determine the ability of test compounds to enhance capillary network formation in growth-limiting conditions. Capillary growth supports angiogenesis, which is critical for development, maturation, and reproductive function. Dysregulation of angiogenesis can lead to birth defects, macular degeneration, impaired wound healing, and tumor growth. This assay measures compound effects on capillary network formation in basal medium, which lacks growth factors that support capillary growth. Tube formation agonists will increase capillary growth in these conditions.

CyteSeer® Data Readout

  • Fluorescence channel #1 DAPI: cell number, ploidy
  • Fluorescence channel #2 (green channel): cell-permeable cytoplasm dye (length, area, and continuity of objects in the capillary network)

Vala Assay: #TUBHMV2

Cell Type: Primary human microvascular-derived endothelial cells (HMVEC-1)

Main Goal of Assay: Determine the ability of test compounds to inhibit capillary network formation in growth-supporting conditions. Capillary growth supports angiogenesis, which is critical for development, maturation, and reproductive function. Dysregulation of angiogenesis can lead to birth defects, macular degeneration, impaired wound healing, and tumor growth. This assay measures compound effects on capillary network formation in complete medium, which contains growth factors that support capillary endothelial cell proliferation. Tube formation antagonists will decrease capillary growth in these conditions.

CyteSeer® Data Readout

  • Fluorescence channel #1 DAPI: cell number, ploidy
  • Fluorescence channel #2 (green channel): cell-permeable cytoplasm dye (length, area, and continuity of objects in the capillary network)

Vala Assay: #TUBIPS1

Cell Type: Human iPS-derived endothelial cells

Main Goal of Assay: Determine the ability of test compounds to enhance blood vessel formation in growth-limiting conditions. Blood vessel growth supports angiogenesis, which is critical for development, maturation, and reproductive function. Dysregulation of angiogenesis can lead to birth defects, macular degeneration, impaired wound healing, and tumor growth. This assay measures compound effects on blood vessel formation in basal medium, which lacks growth factors that support vessel growth. Tube formation agonists will increase vessel growth in these conditions.

CyteSeer® Data Readout

  • Fluorescence channel #1 DAPI: cell number, ploidy
  • Fluorescence channel #2 (green channel): cell-permeable cytoplasm dye (length, area, and continuity of objects in the capillary network)

Vala Assay: #TUBIPS2

Cell Type: Human iPS-derived endothelial cells

Main Goal of Assay: Determine the ability of test compounds to inhibit blood vessel formation in growth-supporting conditions. Blood vessel growth supports angiogenesis, which is critical for development, maturation, and reproductive function. Dysregulation of angiogenesis can lead to birth defects, macular degeneration, impaired wound healing, and tumor growth. This assay measures compound effects on blood vessel formation in complete medium, which contains growth factors that support vascular endothelial cell proliferation. Tube formation antagonists will decrease vessel growth in these conditions.

CyteSeer® Data Readout

  • Fluorescence channel #1 DAPI: cell number, ploidy
  • Fluorescence channel #2 (green channel): cell-permeable cytoplasm dye (length, area, and continuity of objects in the capillary network)

Adipogenesis-Lipolysis Assays

Vala Assay: #A1

Cell Type:  Primary human subcutaneous adipocytes

Main Goal of Assay: Determine test compound effects on adipogenesis in primary human preadipocytes. Fat accumulation is highly correlated with heart disease, fatty liver disease, and atherosclerosis. Reduced adipogenesis contributes to cachexia (wasting disease) associated with cancer and HIV infection. When cultured in media that increases cyclic AMP and glucocorticoid hormones, preadipocytes differentiate into mature adipocytes, which feature large lipid droplets and express adipocyte biomarkers (e.g., ADFP and perilipin). Determining compound effects on adipogenesis can reveal their role in overall health, metabolism, and organ function.

CyteSeer® Data Readout

  • Bright field: rate of lipid droplet growth over 14 to 21 days
  • Fluorescence channel #1 (DAPI): cell number, ploidy
  • Fluorescence channel #2 (green channel, lipid): Lipid droplet count, area, subcellular distribution
  • Fluorescence channel #3 (red channel, perilipin): Expression level and colocalization with the lipid droplets of perilipin, a protein that participates in fat metabolism
  • Fluorescence channel #4 (far red channel, ADFP): Expression level and colocalization with the lipid droplets of ADFP, a protein thought to initiate lipid droplet formation

Vala Assay: #A2

Cell Type:  Primary human subcutaneous adipocytes

Main Goal of Assay: Determine test compound effects on adipogenesis in primary human preadipocytes. Fat accumulation is highly correlated with heart disease, fatty liver disease, and atherosclerosis. Reduced adipogenesis contributes to cachexia (wasting disease) associated with cancer and HIV infection. When cultured in media that increases cyclic AMP and glucocorticoid hormones, preadipocytes differentiate into mature adipocytes, which feature large lipid droplets and express adipocyte biomarkers (e.g., ADFP and perilipin). This version of the adipogenesis assay is performed in the presence of 100 nM rosiglitazone (or a PPAR-γ agonist with similar effects) and is designed to specifically detect adipogenesis inhibitors.

CyteSeer® Data Readout

  • Bright field: rate of lipid droplet growth
  • Fluorescence channel #1 (DAPI): cell number, ploidy
  • Fluorescence channel #2 (green channel, lipid): Lipid droplet count, area, subcellular distribution
  • Fluorescence channel #3 (red channel, perilipin): Expression level and colocalization with the lipid droplets of perilipin, a protein that participates in fat metabolism
  • Fluorescence channel #4 (far red channel, ADFP): Expression level and colocalization with the lipid droplets of ADFP, a protein thought to initiate lipid droplet formation

Vala Assay: #L1

Cell Type:  Primary human subcutaneous adipocytes

Main Goal of Assay: Determine test compound effects on hormone sensitive lipase (HSL) translocation and perilipin phosphorylation in mature human adipocytes. When metabolic energy needs increase, lipid droplets within adipocytes are subject to lipolysis, in which triglycerides are broken down into fatty acids and glycerol. Lipolysis involves the phosphorylation of perilipin, a protein that coats lipid droplets, and the phosphorylation and translocation of HSL to the droplets. Determining compound effects on lipolysis can reveal their role in overall health, metabolism, and organ function.

CyteSeer® Data Readout

  • Fluorescence channel #1 (DAPI): toxicity (cell loss and altered nuclear morphology)
  • Fluorescence channel #2 (green channel): lipid droplets (number, size, and morphology)
  • Fluorescence channel #3 (red channel): phospho-perilipin (expression level and colocalization with lipid droplets)
  • Fluorescence channel #4 (far red channel): phospho-HSL (expression level and colocalization with lipid droplets)

Vala Assay: #L2

Cell Type: Primary human subcutaneous adipocytes

Main Goal of Assay: Determine test compound effects on hormone sensitive lipase (HSL) translocation and perilipin phosphorylation in mature human adipocytes. When metabolic energy needs increase, lipid droplets within adipocytes are subject to lipolysis, in which triglycerides are broken down into fatty acids and glycerol. Lipolysis involves the phosphorylation of perilipin, a protein that coats lipid droplets, and the phosphorylation and translocation of HSL to the droplets. This version of the lipolysis assay is performed in the presence of isoproterenol, which strongly activates HSL and perilipin phosphorylation. Compounds are tested for their ability to inhibit or enhance the isoproterenol response.

CyteSeer® Data Readout

  • Fluorescence channel #1 (DAPI): toxicity (cell loss and altered nuclear morphology)
  • Fluorescence channel #2 (green channel): lipid droplets (number, size, and morphology)
  • Fluorescence channel #3 (red channel): phospho-perilipin (expression level and colocalization with lipid droplets)
  • Fluorescence channel #4 (far red channel): phospho-HSL (expression level and colocalization with lipid droplets)

Vala Assay: #L3

Cell Type: Murine 3T3L1 cell-derived adipocytes

Main Goal of Assay: Determine test compound effects on hormone sensitive lipase (HSL) translocation and perilipin phosphorylation in in murine 3T3L1 adipocytes. When metabolic energy needs increase, lipid droplets within adipocytes are subject to lipolysis, in which triglycerides are broken down into fatty acids and glycerol. Lipolysis involves the phosphorylation of perilipin, a protein that coats lipid droplets, and the phosphorylation and translocation of HSL to the droplets. Because murine and human adipocytes differ in their lipolytic responses to certain agents, this version of the assay reports compound effects specific to murine metabolism.

CyteSeer® Data Readout

  • Fluorescence channel #1 (DAPI): toxicity (cell loss and altered nuclear morphology)
  • Fluorescence channel #2 (green channel): lipid droplets (number, size, and morphology)
  • Fluorescence channel #3 (red channel): phospho-perilipin (expression level and colocalization with lipid droplets)
  • Fluorescence channel #4 (far red channel): phospho-HSL (expression level and colocalization with lipid droplets)

Vala Assay: #LD2

Cell Type: Human HuH-7 cells

Main Goal of Assay: Determine test compound effects on endogenous lipid droplet content in HuH-7 cells. Fatty liver disease is associated with increased lipid droplet formation within hepatocytes. HuH-7 cells are a human hepatocellular carcinoma-derived cell line with a moderate number of endogenous lipid droplets. Compounds that affect triglyceride formation or induce metabolic stress can affect lipid droplet number, size, and/or morphology. This assay also quantifies expression of ADFP, the major lipid droplet-associated protein in hepatocytes.

CyteSeer® Data Readout

  • Fluorescence channel #1 (DAPI): cell number, apoptosis, ploidy
  • Fluorescence channel #2 (green channel): lipid droplets (number, size, and morphology)
  • Fluorescence channel #3 (red channel): ADFP (expression level and colocalization with lipid droplets)

Membrane Protein Assays

Vala Assay: #BC1

Cell Type: HeLa

Main Goal of Assay: Quantify β-catenin expression and cellular localization, particularly in the membrane, nuclear, and cytoplasmic compartments. Under normal conditions, glycogen synthase kinase 3B (GSK3B) phosphorylates β-catenin, leading to its degradation via the ubiquitination/protease system. Wnt pathway activation inhibits GSK3B, reducing β-catenin phosphorylation and degradation. This leads to increased β-catenin expression and β-catenin translocation to the nucleus, where it increases the transcription of proteins that upregulate mitotic activity. Nuclear β-catenin is associated with increased mitosis and can promote tumor formation and growth.

CyteSeer® Data Readout

  • Fluorescence channel #1 DAPI: cell number, ploidy
  • Fluorescence channel #2 (green channel): β-catenin
  • Fluorescence channel #3 (red channel): N-cadherin, E-cadherin, or pan-cadherin

Vala Assay: #NCAD1

Cell Type: N-cadherin expression

Main Goal of Assay:  Quantify test compound effects on N-cadherin expression and cellular localization. Cadherins are cell adhesion molecules that provide a mechanical linkage between cells and support cell differentiation, embryogenesis, and migration. Tumor progression is often associated with an upregulation of N-cadherin, which facilitates tumor cell migration and metastasis. This assay can be multiplexed with pan-cadherin immunolabeling to measure changes in N-cadherin expression relative to other cadherins. This assay can also be multiplexed with the β-catenin assay to provide concurrent information on compound effects on β-catenin expression and distribution.

CyteSeer® Data Readout

  • Fluorescence channel #1 (DAPl): cell number, ploidy
  • Fluorescence channel #2 (green channel): N-Cadherin
  • Fluorescence channel #3 (red channel): pan-cadherin or β-catenin

Vala Assay: #ECAD1

Cell Type: Human A431 cells, derived from epithelial carcinoma

Main Goal of Assay: Quantify test compound effects on E-cadherin expression and cellular localization. Cadherins are cell adhesion molecules that provide a mechanical linkage between cells and support cell differentiation, embryogenesis, and migration. E-cadherin levels regulate cell division, and loss of E-cadherin leads to release of β-catenin, Wnt signaling activation, transcription of proto-oncogenes, and tumorigenesis. This assay can be multiplexed with pan-cadherin immunolabeling to measure changes in E-cadherin expression relative to other cadherins. This assay can also be multiplexed with the β-catenin assay to provide concurrent information on compound effects on β-catenin expression and distribution.

CyteSeer® Data Readout

  • Fluorescence channel #1 (DAPl): cell number, ploidy
  • Fluorescence channel #2 (green channel): E-Cadherin
  • Fluorescence channel #3 (red channel): pan-cadherin or β-catenin

Vala Assay: #VCAD1

Cell Type: Primary human microvascular endothelial cells

Main Goal of Assay: Quantify test compound effects on VE-cadherin expression and cellular localization. VE-cadherin, the major adherens junction protein in vascular endothelial cells, regulates cell-cell adhesion, angiogenesis, and vascular permeability. VE-cadherin contributes to tumor-induced angiogenesis and transendothelial migration of metastatic cancer cells. This assay can be multiplexed with pan-cadherin immunolabeling to measure changes in VE-cadherin expression relative to other cadherins. This assay can also be multiplexed with the β-catenin assay to provide concurrent information on compound effects on β-catenin expression and distribution.

CyteSeer® Data Readout

  • Fluorescence channel #1 (DAPl): cell number, ploidy
  • Fluorescence channel #2 (green channel): VE-Cadherin
  • Fluorescence channel #3 (red channel): pan-cadherin or β-catenin

Vala Assay: #ZOOTJP1

Cell Type: Canine MDCK cells, derived from kidney epithelial tissue

Main Goal of Assay: Determine test compound effects on ZO-1 expression and localization. Zonula occuldens protein 1 (ZO-1) is a key component of tight junctions, which control the structure, function, and permeability of epithelial and endothelial cell sheets. ZO-1 also negatively regulates epithelial cell proliferation and the epithelial-mesenchymal transition (EMT). This assay therefore reports compound effects on tight junction and epi/endothelial integrity. This assay can detect either upregulation or downregulation of ZO-1 in MDCK cells.

CyteSeer® Data Readout

  • Fluorescence channel #1 DAPI: cell number, ploidy
  • Fluorescence channel #2 (green channel): ZO-1

Mitochondrial Function Assays

Vala Assay: #MP1

Cell Type: Human HuH-7 cells, derived from hepatocellular carcinoma

Main Goal of Assay:  Assess mitochondrial and cellular health by evaluating mitochondrial membrane potential after 30, 60, and 90 minutes of exposure to test compounds. During apoptosis, mitochondria lose their membrane potential due to collapse of the H+ gradient across the inner mitochondrial membrane. Tetramethyl rhodamine methyl ester (TMRM) is a cell permeant cationic dye that accumulates in the matrix of healthy mitochondria due to the negative membrane potential. When the membrane potential is lost, TMRM uptake into mitochondria is prevented and the fluorescent signal is lost.

CyteSeer® Data Readout

  • Fluorescence channel #1 (Hoescht): cell number, nuclear size, ploidy, apoptotic fragmentation
  • Fluorescence channel #2 (red channel): TMRM (mitochondrial membrane potential)

Vala Assay: #MP2

Cell Type: Human HuH-7 cells, derived from hepatocellular carcinoma

Main Goal of Assay: Assess mitochondrial and cellular health by evaluating mitochondrial membrane potential after 72 hours of exposure to test compounds. During apoptosis, mitochondria lose their membrane potential due to collapse of the H+ gradient across the inner mitochondrial membrane. Tetramethyl rhodamine methyl ester (TMRM) is a cell permeant cationic dye that accumulates in the matrix of healthy mitochondria due to the negative membrane potential. When the membrane potential is lost, TMRM uptake into mitochondria is prevented and the fluorescent signal is lost.

CyteSeer® Data Readout

  • Fluorescence channel #1 (Hoescht): cell number, nuclear size, ploidy, apoptotic fragmentation
  • Fluorescence channel #2 (red channel): TMRM (mitochondrial membrane potential)

Differentiation Assays

Vala Assay: #PBCD1

Cell Type: T6PNE cells, derived from human fetal islets

Main Goal of Assay: Determine test compound effects on human pancreatic beta-cell differentiation using insulin gene promoter-driven GFP expression as a readout. This assay uses T6PNE, a cell line derived from human fetal islets and engineered to express a tamoxifen-inducible form of the E47 transcription factor and eGFP under control of the human insulin promoter. Forty-eight-hour tamoxifen treatment induces global gene expression changes consistent with beta-cell differentiation, including 100-fold upregulation of insulin gene expression. Compounds that affect beta-cell differentiation or function will affect insulin-driven eGFP expression in T6PNE cells.

CyteSeer® Data Readout

  • Fluorescence channel #1 (DAPI): cell number, ploidy
  • Fluorescence channel #2 (green channel): GFP as a reporter of insulin gene expression per cell

Kinase Activation Assays

Vala Assay: #P38MPK1

Cell Type: HeLa

Main Goal of Assay: Determine test compound effects on phosphorylation and cellular localization of p38-MAPK (mitogen-activated protein kinase). p38-MAPK activation occurs in response to cell stressors such as oxidative stress, inflammation, ultraviolet radiation, and heat shock. p38-MAPK also participates in cell differentiation, proliferation, tumorigenesis, and metastasis. In response to stimuli, p38-MAPK translocates to the nucleus, where it phosphorylates transcription factors such as FOXO1. This assay provides an integrated readout of compound effects on p38-MAPK-activating signaling pathways within cells. This assay can be multiplexed to measure co-incident activation of another kinase along with p38-MAPK.

CyteSeer® Data Readout

  • Fluorescence channel #1 DAPI: cell number, ploidy
  • Fluorescence channel #2 (green channel): phospho-p38-MAPK (expression level and nuclear localization)
  • Fluorescence channel #3 (red channel): multiplexed staining for another phosphorylated kinase (optional)

Vala Assay: #ERKMPK1

Cell Type: HeLa

Main Goal of Assay: Determine test compound effects on phosphorylation and cellular localization of ERK-MAPK (mitogen-activated protein kinase). ERK-MAPK plays key roles in embryonic development, cardiac function, cell proliferation, and tumorigenesis. In response to stimuli, ERK-MAPK translocates to the nucleus, where it phosphorylates transcription factors such as ELK. This assay provides an integrated readout of compound effects on ERK-MAPK-activating signaling pathways within cells. This assay can be multiplexed to measure co-incident activation of another kinase along with ERK-MAPK.

CyteSeer® Data Readout

  • Fluorescence channel #1 DAPI: cell number, ploidy
  • Fluorescence channel #2 (green channel): phospho-ERK-MAPK (expression level and nuclear localization)
  • Fluorescence channel #3 (red channel): multiplexed staining for another phosphorylated kinase (optional)

Vala Assay: #JNKMPK1

Cell Type: HeLa

Main Goal of Assay: Determine test compound effects on phosphorylation and cellular localization of JNK-MAPK (mitogen-activated protein kinase). JNK-MAPK activation occurs in response to cell stressors such as oxidative stress, inflammation, ultraviolet radiation, and heat shock. JNK-MAPK also participates in cell differentiation, proliferation, tumorigenesis, and metastasis. In response to stimuli, JNK-MAPK translocates to the nucleus, where it phosphorylates transcription factors such as c-Jun. This assay provides an integrated readout of compound effects on JNK-MAPK-activating signaling pathways within cells. This assay can be multiplexed to measure co-incident activation of another kinase along with JNK-MAPK.

CyteSeer® Data Readout

  • Fluorescence channel #1 DAPI: cell number, ploidy
  • Fluorescence channel #2 (green channel): phospho-JNK-MAPK (expression level and nuclear localization)
  • Fluorescence channel #3 (red channel): multiplexed staining for another phosphorylated kinase (optional)

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