#WHAT IS CCCP TREATMENT MANUAL#
* Please consult the product manual or contact technical service for additional information on the fixability of these reagents. The ready-to-use CellLight® reagent is added to live cells, followed by an overnight incubation to allow for protein expression.Ĭells are fixed and permeabilized, incubated with the antibody for labeling, and visualized with a fluorescently labeled secondary antibody.** Dye-specific working concentrations and incubation times are provided in the product’s manual. Incubate cells with the reagent for approximately 5–30 min. Compatible with MitoTracker® and CellLight® reagents. Live-cell imaging applications fixable,*† thus compatible with antibody-based imaging applications. Live-cell imaging applications some reagents are fixable,* thus compatible with antibody-based imaging applications.
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Recognize specific protein target of interest (e.g., anti–OxPhos complex IV). Positively charged dyes localize to actively respiring mitochondria.Ĭombine targeting sequence–fluorescent protein fusion with the transduction efficiency of BacMam to label organelles independently of function (i.e., pH, mitochondrial membrane potential).
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(e.g., MitoTracker®, JC-1, and TMRM dyes) Fluorescent detection reagents for visualizing mitochondria are summarized in Table 1. Like conventional stains, the MitoTracker® probes are sequestered by functioning mitochondria however, cells stained with these dyes retain their fluorescent staining patterns during subsequent fixation and permeabilization steps. MitoTracker® Green FM, Orange CMTMRos, Red CMXRos, and Deep Red FM probes overcome these limitations. Conventional stains such as rhodamine 123 and tetramethylrhodamine methyl ester (TMRM) are not maintained in the mitochondrion once its membrane potential is lost, limiting their use in experiments in which cells are subsequently fixed and permeabilized or treated with agents that affect their energetic state. These dyes thereby enable researchers to probe mitochondrial activity, localization, and abundance, as well as to monitor changes in membrane potential in response to pharmacological agents that alter mitochondrial function. The uptake of mitochondrion-selective dyes is dependent on the mitochondrial membrane potential. We also offer a range of mitochondrion-selective dyes with which to monitor mitochondrial morphology and function.
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BacMam reagents enable titratable and reproducible expression and high cotransduction efficiency, and they are compatible with many cell types, including those traditionally difficult to transfect such as primary neurons and stem cells. Useful for both live-cell imaging and fixed-cell analyses, these CellLight® reagents are prepackaged BacMam expression vectors encoding a fluorescent protein (GFP or RFP) fused to the leader sequence of the mitochondrial protein E1α–pyruvate dehydrogenase. These OxPhos-specific antibodies are versatile tools for investigating mitochondrial morphogenesis, as well as for characterizing degenerative diseases such as Alzheimer’s disease and Parkinson’s disease that are associated with defects in mitochondrial function.Īs with the OxPhos-specific antibodies, the CellLight® Mitochondria-GFP and -RFP reagents localize to the mitochondria independent of mitochondrial membrane potential (Figures 2 and 3). We offer a number of well-characterized antibodies against different proteins and epitopes within the OxPhos complexes (Figure 1). The simple act of staining mitochondria and visualizing their morphology can provide a significant amount of information regarding their overall biology and functional state.Īntibodies to the oxidative phosphorylation (OxPhos) complexes can be used to visualize mitochondria when used in conjunction with fluorescent secondary antibodies. The number of mitochondria is a function of several variables, including cell type, cell-cycle and differentiation stage, cellular energy level, and overall cell health. This organelle can switch between a fragmented morphology, with many ovoid-shaped mitochondria, and a reticulum, in which the mitochondrion is a single, many-branched structure. Mitochondrial morphology is highly variable.