Key features and details
- Detection method: Fluorescent
- Platform: Flow cytometer
- Sample type: Adherent cells, Suspension cells
Product nameMitoBiogenesis™ Flow Cytometry Kit
Sample typeAdherent cells, Suspension cells
Species reactivityReacts with: Mouse, Rat, Cow, Human, Caenorhabditis elegans
Abcam’s MitoBiogenesis™ Flow Cytometry kit is designed to evaluate drug-induced effects on mitochondrial biogenesis early in the safety screening process. The assay combines the power of single cell analysis obtained with flow cytometry to evaluate the ratio between two important mitochondrial proteins, one encoded by the mitochondrial DNA (mtDNA) and one encoded by the nuclear DNA (nDNA).
The two proteins are each subunits of a different oxidative phosphorylation enzyme complex, one protein being subunit I of Complex IV (COX-I), which is mtDNA-encoded, and the other being the 70 kDa subunit of Complex II (SDH-A), which is nDNA-encoded. Complex IV includes several proteins which are encoded in the mitochondrion, while the proteins of Complex II are entirely encoded in the nucleus.
Human, rat and mouse cells are treated for several doublings then are harvested, fixed and permeabilized in suspension. The in-vivo levels of the target proteins are detected simultaneously in each cell with highly specific, well-characterized, monoclonal antibodies that are labeled with Alexa® fluorophores. Thus, minimizing potential changes during sample preparation and handling, such as preparation of protein extracts.
The Alexa Fluor® dye included in this product is provided under an intellectual property license from Life Technologies Corporation. As this product contains the Alexa Fluor® dye, the purchase of this product conveys to the buyer the non-transferable right to use the purchased product and components of the product only in research conducted by the buyer (whether the buyer is an academic or for-profit entity). As this product contains the Alexa Fluor® dye the sale of this product is expressly conditioned on the buyer not using the product or its components, or any materials made using the product or its components, in any activity to generate revenue, which may include, but is not limited to use of the product or its components: (i) in manufacturing; (ii) to provide a service, information, or data in return for payment (iii) for therapeutic, diagnostic or prophylactic purposes; or (iv) for resale, regardless of whether they are sold for use in research. For information on purchasing a license to use products containing Alexa Fluor® dyes for purposes other than research, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or firstname.lastname@example.org
Review the mitochondrial assay guide, or the full metabolism assay guide to learn about more assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also how to assay metabolic function in live cells using your plate reader.
Storage instructionsStore at +4°C. Please refer to protocols.
Components 100 tests 100x MTCO1 AlexaFluor® 488 + SDHA AlexaFluor® 647 Cocktail 1 x 100µl 10X Blocking Buffer 1 x 6ml 10X Phosphate Buffered Saline 1 x 100ml
- Cyclooxygenase 1
A total of 10,000 gated events were captured for analysis of HeLa cells with or without chloramphenicol (16 µM, 6-days). The results show MTCO1 Alexa®488, FL1(A) and SDHA Alexa®647, FL4(B) co staining for untreated, no anitobdies (blue); untreated with antibodies (black); chloramphenicol, no antibodies (purple); and chloramphenicol with antibodies (red).
HeLa cells were treated with 2-fold serial dilution of chloramphenicol for 6 days. The relative levels of the mean FL-1 fluorescence (MTCO1 Alexa® 488) and FL-4 fluorescence (SDHA Alexa® 647); a total of 10,000 events were collected.
Cells were treated with 30 µM chloramphenicol for a period fo 6 days. Results of immunostaining of HeLa cells showing MTCO1 Alexa® 488 antibody (ab154477, green) on untreated (A) and chloramphenicol treated (D). SDHA Alexa® 647 antibody (ab168536, red) on untreated (B) and chloramphenicol treated (E). Merge of color channels for untreated (C) and chloramphenicol treated (F) cells.
A total of 10 µg from Human or rat cultured cells were probed with the primary and secondary antibodies and scanned with a LI-COR® Odyssey® imager. Reduction of mtDNA levels in human Rho0 (mtDNA depleted) cells, or inhibition of mitochondrial protein translation by chloramphenicol in rat cells result in specific reduction of COX-I protein while nuclear DNA-encoded SDH-A is unaffected.
ab168540 has not yet been referenced specifically in any publications.