TempoMito™

Mitochondrial Biosensor Assay

TempoMito™

Mitochondrial Biosensor Assay

TempoMito™ mitochondrial calcium biosensor responds as a fluorescent reporter: 605nm-625nm fluorescent range. It is incorporated into Tempo’s proprietary immortalized human neuroepithelial cell line, TempoRapidTM. TempoMito™ control cells serve as a control for all products containing the TempoMito™ biosensor. This product can be used for single-cell applications, as well as medium to high-throughput screening platforms.

TempoMito™-HEK293t

TempoMito™-HEK293t Cell Line

HEK293t cell line is one of the most widely used human immortalized cell lines in research and drug development. The cells are very easy to work with and can perform a variety of functional applications (e.g., protein purification, expression studies, differential screening, functional reporter assays, receptor-ligand assays, etc).

TempoMito™ biosensor is an mitochondrial calcium flux sensor. It is introduced to the cells using proprietary liposome mediated methods and is expressed in the mitochondria (using marker proteins such as COX IV). HEK293t stably expressing TempoMito™ biosensor has been developed for monitoring mitochondrial calcium flux and cytotoxicity using a highly sensitive fluorescent based biosensor as reporter.

Culture Medium: DMEM with 2mM L-Glutamine; 10% Fetal Bovine Serum (FBS). Subculture Routine: Split sub-confluent using 0.025% trypsin/EDTA solution. Allow the flask to sit at 37°C until the cells detach. Add fresh medium, aspirate and dispense into new culture flasks. Passage ratio: 1-to-9.

TempoMito™-U2OS

TempoMito™-U2OS Cell Line

U2-OS cell line, originally known as the 2T cell line, is an immortalized human cancer cell line. It is widely used in research and drug development, as it offers a variety of application potentials.

TempoMito™ biosensor is an mitochondrial calcium flux sensor. It is introduced to the cells using proprietary liposome mediated methods and is expressed in the mitochondria (using marker proteins such as COX IV). U2-OS stably expressing TempoMito™ biosensor has been developed for monitoring mitochondrial calcium flux and cytotoxicity using a highly sensitive fluorescent based biosensor as reporter.

Culture Medium: McCoy′s 5a medium with 1.5 mM Glutamine or DMEM with 2mM L-Glutamine; 10% Fetal Bovine Serum (FBS). Subculture Routine: Split sub-confluent using 0.025% trypsin/EDTA solution. Allow the flask to sit at 37°C until the cells detach. Add fresh medium, aspirate and dispense into new culture flasks. Passage ratio: 1-to-6

TempoMito™ + Tempo-iAstro™

TempoMito™ Mitochondrial Biosensor Assay using Human Astrocytes

TempoMito™-iAstro cells contain mitochondrial biosensors in human iPS-derived astrocytes. Disruption of mitochondrial functions in human astrocytes has been implicated in Alzheimer’s disorder and ischemia. Tempo’s biosensor responds to mitochondrial calcium changes and reports in the 605nm-625nm fluorescent range (Excitation/Emission) as calcium levels increase or decrease. With excellent signal-to-noise ratios, these sensors can operate on second-to-minute scales. This tool provides an efficient way of detecting changes in the mitochondrial organelle and enables high-throughput screening approaches for drug discovery and toxicology screening.

TempoMito™-iAstro: Mitochondrial Calcium Biosensor Assay using human Astrocytes

TempoMito™-iAstro are derived from integration-free induced pluripotent stem cell (iPSC) lines under a fully defined proprietary astrocytes induction condition. TempoMito™-iAstro cells are plated as monolayers in culture and express markers such as GFAP. TempoMito™ biosensor is introduced to the cells using proprietary liposome mediated methods and is expressed in the mitochondria (using marker proteins such as COX IV).

TempoMito™ + Tempo iNStem™

TempoMito™ Mitochondrial Biosensor Assay using Human Neural Progenitors

TempoMito™-iNStem cells comprise mitochondrial biosensors incorporated into human iPS-derived neural progenitor cells. Mitochondrial dysfunctions have been implicated in human neurological and psychiatric disorders, and neural progenitor cells have been used as a cellular model. Tempo’s biosensor responds to mitochondrial calcium changes and reports in the 605nm-625nm fluorescent range (Excitation/Emission) as calcium levels increase or decrease. With excellent signal-to-noise ratios, these sensors can operate on second-to-minute scales. This tool provides an efficient way of detecting changes in the mitochondrial organelle and enables high-throughput screening approaches for drug discovery and toxicology screening.

TempoMito™-iNStem;: Mitochondrial Calcium Biosensor Assay using human Neural Stem Cells. They are derived from integration-free induced pluripotent stem cell (iPSC) lines under a fully defined proprietary neural induction condition. TempoMito™-iNStem cells are polarized structures when plated as a monolayer in culture and express markers such as Vimentin. We’ve validated iNStem™ cells to be multipotent neural and glial progenitor cells, as they can be further differentiated into neural and glial cell types under specific in vitro reprogramming conditions. TempoMito biosensor is introduced to the cells using proprietary liposome mediated methods and is expressed in the mitochondria (using marker proteins such as COX IV).

TempoMito™ + TempoRapid™

TempoMito™ Mitochondrial Biosensor Assay using Immortalized Human Neuroepithelial Cells

TempoMito™ mitochondrial calcium biosensor responds as a fluorescent reporter: 605nm-625nm fluorescent range. It is incorporated into Tempo’s proprietary immortalized human neuroepithelial cell line, TempoRapidTM. TempoMito™ control cells serve as a control for all products containing the TempoMito™ biosensor. This product can be used for single-cell applications, as well as medium to high-throughput screening platforms.

Confocal image of TempoMito™ in Astrocytes

Immunocytochemistry with anti-COX IV antibodies

TempoMito™ -control cells

TempoMito™-iNStem

Neural Progenitor cells, ICC, Vimentin marker

TempoMito™ with stimulation

TempoMito™ responses

TempoMito™ responses

TempoMito™ responses

TempoCal™ and TempoMito™

TempoMito™-Rapid: Mitochondrial Biosensor Assay using Immortalized Human Neuroepithelial Cells

Applications

TempoATP™-TempoMito™ incorporated cells are intended for research and development, compound discovery and therapeutics development use only. It is not a product for human testing or diagnostics.


Neurotoxicity testing for reporting cellular calcium fluctuations


Phenotypic Assays (i.e., mitochondrial toxicity and function)


Chemical compound or small molecules testing


Cell morphology or cell death research studies


High Content Assays


Live Cell Imaging


Research and development on animal models for human diseases


Studying neural maturation, networking and function, and cations-mediated pathways


Mitochondrial and Cellular metabolism


Receptor studies (e.g., IGF-I, IGF-II)

Specifications

~1×10^6 cells per 1ml of freezing medium (vial)

Long-term Storage: liquid nitrogen

Growth Properties: adherent

Technology used: an in-house developed proprietary feeder-free, serum-free, viral/genetic-elements-free, integration-free reprogramming.

QC: Sterility, Safety (BioSafety Level 2), HIV/viruses, bacteria, fungi: negative. Cell viability post-thawing (>90%)

TempoMito™-HEK293t SKU302
1 vial = ~0.5×10^6 cells

TempoMito™-U2OS SKU301
1 vial = ~0.5×10^6 cells

TempoMito™-iAstro SKU103

TempoMito™-iNStem SKU107
Storage: remove cryovials (dry ice packaging) and place the vial into liquid nitrogen for storage. Alternatively, thaw and use the cells immediately.

TempoMito™-Rapid SKU109
Storage: remove cryovials (dry ice packaging) and place the vial into liquid nitrogen for storage. Alternatively, thaw and use the cells immediately.
Starting Materials
: immortalized human cell line of neuro-epithelial origin. Cells should grow fairly well (doubling time <36 h) without passage limitation in a conventional medium DMEM or DMEM/F12 media with a maximum of 10% serum and selection antibiotics (G418, hygromycin or puromycin) at reasonable concentrations.

Frequently Requested Cell Lines
The following are disease cell models that we are able to create through TempoMito™-HEK293t and TempoMito™-U2OS

Cell Line Name Histology Type
U2OS Bone carcinoma
HEK293 Immortalized Kidney Epithelial
SW 480 Colorectal adenocarcinoma
U87MG Brain cancer
Hep G2 Hepatocellular carcinoma
NIH3T3 Embroyonic fibroblast, murine
HT1080 Carcinoma (connective tissue)
HCT116 Colorectal cancer
MCF 7 Breast cancer
SW 1990 Pancreatic cancer
NCI-H2126 Non-Small cell cancer
HUVEC Umbilical vascular endothelium
HeLa Cervical epithelial adenocarcinoma
CG4 Glial cell line (from rat)
A-204 Muscle epithelial
A549 Lung epithelial cell line
BT-142 Oligoastrocytoma, suspension
NK-92 Peripheral blood, natural killer cells
CHO Chinese hamster ovarian cell line
NCI-H69 Lung tumor cell aggregate, suspension
ARPE-19 Retinal pigmented epithelial
Beta-TC-6 Beta cell (mouse)
BxPC-3 Pancreatic epithelial, adenocarcinoma
Jurkat cells Peripheral T lymphocyte, suspension
SW 780 Urinary bladder epithelial carcinoma
MeT-5A Mesothelium, from non-cancerous
NFKB reporters Email us
Mesenchymal stromal cells Email us
Chemokine Reporter cells Email us
cell line of your choice Email us

References

TempoMito™-HEK293t

  • Dykens JA, Jamieson JD, Marroquin LD, Nadanaciva S, Xu JJ, Dunn MC, Smith AR, Will Y. Toxicol Sci. 2008 Jun;103(2):335-45.
  • Dykens JA, Will Y. Drug Discov today. 2007 Sep; 12 (17-18): 777-85.

TempoMito™-iAstro

  • Martin LJ. Prog Mol Biol Transl Sci. 2012;107:355-415.
  • Martin LJ. Pharmaceuticals (Basel). 2010;3(4):839-915.
  • Chen K, Northington FJ, Martin LJ. Brain Struct Funct. 2010 Mar;214(2-3):219-34.
  • Palomo GM, Manfredi G. Brain Res. 2015 May 14;1607:36-46.
  • Dykens JA, Jamieson JD, Marroquin LD, Nadanaciva S, Xu JJ, Dunn MC, Smith AR, Will Y. Toxicol Sci. 2008 Jun;103(2):335-45.
  • Dykens JA, Will Y. Drug Discov today. 2007 Sep; 12 (17-18): 777-85.

TempoMito™-iNStem

  • Martin LJ. Prog Mol Biol Transl Sci. 2012;107:355-415.
  • Martin LJ. Pharmaceuticals (Basel). 2010;3(4):839-915.
  • Chen K, Northington FJ, Martin LJ. Brain Struct Funct. 2010 Mar;214(2-3):219-34.
  • Palomo GM, Manfredi G. Brain Res. 2015 May 14;1607:36-46.
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