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Mitochondria Nucleus Lysosomes Golgi ER
Mitochondria are regarded as the 'powerhouse' of the cell. These double-membrane bound organelles produce a bulk of the cells energy in the form of adenosine triphosphate (ATP) via cellular respiration. They play key roles in several cellular processes including cell signaling, cellular differentiation and apoptosis; and their dysfunction have been linked to disorders such as Alzheimer's disease and cancer. Live cell mitochondrial imaging is typically performed using organelle stains such as MitoLite™dyes, membrane potential probes such as JC-1 or JC-10™, and reactive oxygen species indicators such as MitoROS™ 580.
Left: Live HeLa cells stained with MitoLite™ Red FX600 (red), ER Green™ (green) and DAPI (blue). Center: Campotothecin-induced mitochondria membrane potential changes were measured with JC-10™ and JC-1 in Jurkat cells. Right: RAW 264.7 cells were treated with PMA (phorbol 12-myristate 13-acetate) to induce hydroxyl radical formation, then stained with MitoROS™ 580 (red) and Nuclear Green™ LCS1 (green).
The cell nucleus, which is a membrane-bound organelle found in eukaryotes, houses a majority of the cell's genetic material in the form of DNA. It is comprised of the nuclear envelope, a double-membrane that encloses the entire organelle and isolates its contents from the cellular cytoplasm, and the nuclear matrix, a network within the nucleus that adds mechanical support. Its primary function is to control gene expression, regulate DNA replication, and preserve the integrity of the DNA. During apoptosis, the nucleus and its contents undergo distinct morphological changes including nuclear condensation and DNA fragmentation. The latter (DNA fragmentation) is regarded as a biochemical hallmark of apoptosis and can be identified using the TUNEL assay. Imaging the nucleus using membrane permeant and impermeant nuclear dyes is commonly used to differentiate live and dead cells. This is based on the premise that membrane-impermeant dyes can freely enter the compromised membranes of dead cells but cannot penetrate the intact membrane of live cells.
Left: Live HeLa cells stained with Nuclear Green™ LCS1 (green) and Annexin-V iFluor® 555 (red) at 37°C for 30 minutes, then washed and imaged. Center: Formaldehyde fixed HeLa cells stained with Nuclear Orange™ DCS1 (orange) and Phalloidin-iFluor® 350 conjugate (blue). Right: Formalin-fixed paraffin-embedded (FFPE) human lung adenocarcinoma stained with Cell Meter™ TUNEL Apoptosis assay (green) and Nuclear Blue™ DCS1 (blue).
Lysosomes are the "waste-disposal" system of the cell digesting unwanted materials and cellular debris in the cytoplasm. These membrane-bound organelles can vary in size from 0.1 to 1.2 µm and maintains an acidic interior (pH ~4.8) via proton pumps embedded in the lysosomal membrane. Lysosomes house various hydrolytic enzymes responsible for digesting biopolymers such as proteins, peptides, nucleic acids, carbohydrates and lipids. Other then digesting biopolymers, lysosomes are involved in cellular processes such as secretion, cell signaling, energy metabolism, autophagy and apoptosis. The synthesis of lysosomal hydrolases are controlled by nuclear genes, and mutations in these genes can cause an array of inherited metabolic disorders. Defects to lysosomal hydrolytic enzymes result in the accumulation of macromolecules or monomeric compounds contributing to abnormal signaling pathways which ultimately lead to pathogenic disorders. Imaging lysosomes can be done using LysoBrite™ dyes, which selectively accumulate inside the lysosomes of live cells via the lysosomal pH gradient.
The Golgi apparatus is a complex of vesicles and folded membranes within the cytoplasm of most eukaryotic cells. It plays an essential role in processing macromolecules from the endoplasmic reticulum (ER) for secretion, the vesicular transport and trafficking of macromolecules for use by other organelles within the cell, and the formation of lysosomes. Dysfunction of the Golgi apparatus has been linked to various neurodegenerative diseases and autosomal recessive disorders. Imaging the Golgi apparatus in live cells can be done using ceramides. Two common ceramides, NBD ceramide and TMR ceramide, have been extensively used to study lipid metabolism and trafficking.
Left: Live HeLa cells stained with Cell Navigator® NBD Ceramide Golgi Staining Kit (green) and Hoechst 33342 (blue) at 37°C for 20 minutes, then washed and imaged. Right:Live HeLa cells stained with Cell Navigator® TMR Ceramide Golgi Staining Kit (green) and Hoechst 33342 (blue) at 37°C for 20 minutes, then washed and imaged.
The endoplasmic reticulum (ER) is an organelle present in most eukaryotic cells that is responsible for the synthesis and transport of cellular proteins, lipids and other biomolecules. Structurally, the ER is comprised of an interconnected network of flattened, membrane-enclosed sacs and tubules that extend from the nuclear membrane throughout the cytoplasm, which are held together and supported by the cytoskeleton. The ER complex can be divided into two subunits, the rough ER and the smooth SER. The surface of the rough ER is coated with protein-manufacturing ribosomes. Once synthesized, membrane-bound transport vesicles shuttle these proteins to the Golgi apparatus. The smooth ER, however, lacks ribosomes and is responsible for the synthesis of lipids, phospholipids and steroids. Imaging the ER in living cells can be done using membrane-permeant ER Tracer™ dyes from our Cell Navigator® Live Cell Endoplasmic Reticulum Staining Kits. These dyes can be multiplexed with other proteins or probes in live cell multiparametric studies or after fixation for colocalization studies.
Left: Live HeLa cells were stained with ER Tracer™ Blue and Nuclear Red™ LCS1. Center: Live HeLa cells were stained with ER Tracer™ Green, MitoLite™ Red FX600 and Hoechst 33342 (blue). Right: Live HeLa cells were stained with ER Tracer™ Red, then formaldehyde-fixed and stained with Phalloidin-iFluor® 488 (green) and DAPI (blue).