Lab Protocols

Apoptosis or programmed cell death is defined as " a mechanism of cellular suicide which occurs after sufficient cellular damage ". Apoptosis is first characterized by a change in the refractive index of the cell followed by cytoplasmic shrinkage and nuclear condensation. The cell membrane begins to show blebs and eventually these blebs separate from the dying cell and form "apoptotic bodies". Apoptotic cells also cease to maintain phospholipid asymmetry in the cell membrane, and phosphotidylserine appears on the outer leaflet. The mitochondrial outer membrane also undergoes changes that include loss of its electrochemical gradient, and substances like cytochrome c leak into the cytoplasm. Finally, adjacent cells or macrophages phagocytose apoptotic bodies and the dying cell. The apoptotic cell does not provoke an inflammatory response, and only individual cells are affectedby apoptosis in vivo . The mechanisms of apoptosis Apoptosis can be induced in response to ...

Procedure: Pellet cells Lyse cells in 0.5 ml detergent buffer: 10 mm Tris (pH 7.4), 5 mm EDTA, 0.2% Triton  Vortex  Incubate on ice for 30 min. Centrifuge at 27000 g for 30 minutes  Divide supernatants into 2-250 μl aliquots  Add 50 ul ice cold 5 M NaCl to each followed by vortexing  Precipitate DNA: Add 0.6 ml 100% EtOH and 150 μl 3 M Na acetate, pH 5.2 place at –80oC freezer for 1 hr. Centrifuge 20,000 g for 20 min and pool DNA extracts by re-dissolving by adding 400 μl total) of extraction buffer (10 mM Tris and 5 mm EDTA).  Add 2 μl (10 mg/ml) DNase free RNase. Incubate for 5 hr at 37 o C.  Add 25 μl Proteinase K at 20 mg/ml and 40 μl of buffer (100 mm Tris, pH 8.0, 100 mM EDTA, 250 mM NaCl. Incubate overnight at 65 o C.  Extract DNA with phenol, choloform, isoamyl alcohol and precipitate with EtOH. From Kotamraju et al JBC, 2000 DNA Fragmentation: A distinctive feature of apoptosis at the biochemical level is DNA fragmentation. T...

Introduction Caspases are essential in cells for apoptosis, one of the main types of programmed cell death in development and most other stages of adult life, and have been termed "executioner" proteins for their roles in the cell. Caspases were first implicated in apoptosis when CED-3, a protein required for programmed cell death in Caenorhabditis elegans, was found to have close homology with the mammalian interleukin-1 -converting enzyme (ICE or caspase 1) and that over-expression of ICE induced apoptosis. Failure of apoptosis is one of the main causes of tumour development and autoimmune diseases. This coupled with the unwanted apoptosis that occurs with ischaemia or Alzheimer's disease, has raised interest in caspases as potential therapeutic targets. Caspases are enzymes known as proteases, which play essential roles in apoptosis and inflammation. As proteases, they are enzymes that cleave other proteins. They are called cysteine proteases, because they use a cyste...

Apoptosis may be induced in experimental systems through a variety of methods. In general, they can be divided into 2 categories: a) biological induction; and b) chemical induction. A) Biological induction of apoptosis Activation of either Fas or TNF-receptors by the respective ligands or by cross-linking with agonist antibody induces apoptosis of Fas- or TNF receptor-bearing cells. Below is general protocol used to induce apoptosis using anti-Fas receptor (CD95) mAb in Jurkat Cells. 1. Grow Jurkat cells in RPMI-1640 medium containing 10% fetal bovine serum in a humidified, 5% CO 2 incubator at 37°C. 2. Suspend the cells in fresh medium at a concentration of 1 × 105 cells/ml. After two to three days of incubation in a 37°C, 5% CO 2 incubator, harvest the cells by centrifugation at 300–350 × g for 5 mins. 3. Resuspend cells in fresh medium to 5 × 105 cells/ml and add CD95 mAb to a final concentration of 0.05 – 0.1 μg/ml. Incubate for 3–6 hours in a 37°C incubator. As a negativ...

The appearance of phosphatidylserine (PS) residues (normally hidden within the plasma membrane) on the surface of the cell is an early parameter of apoptosis, which can also be used to detect and measure apoptosis. During apoptosis, PS is translocated from the cytoplasmic face of the plasma membrane to the cell surface. Annexin V has a strong, Ca2+-dependent affinity for PS and therefore can be used as a probe for detecting apoptosis. Example protocol -  Protocol for Annexin V-FITC Apoptosis Detection Kit (ab14085) : A) Incubation of cells with Annexin V-FITC Induce apoptosis by desired method. Collect 1-5 x 10 5 cells by centrifugation. Resuspend cells in 500 μl of 1X Binding Buffer. Add 5 μl of Annexin V-FITC and 5 μl of propidium iodide (PI, optional.) Incubate at room temperature for 5 min in the dark. Proceed to B or C below depending on method of analysis. B) Quantification by flow cytometry Analyze Annexin V-FITC binding by flow cytometry (Ex = 488 nm; Em = 530 nm) ...

Abstract In an earlier article from this laboratory, the current methods developed to detect apoptosis in cells and tissues were highlighted, along with the challenges in their interpretation. Recent discoveries concerning the underlying biochemical mechanisms of apoptotic effector pathways have made possible further assays that allow a more direct measure of the activation of the apoptotic machinery in cells. This article summarizes some of these newer methods and extends the interpretation of the more classical assays of apoptosis in a defined cell system. We present data in KB and PC3 cell model culture systems induced to undergo apoptosis by the plant toxin ricin. Using a modified in situ nick translation assay (ISNT) with either Bodipy or BUdR labeling, we confirm that most cells showing altered nuclear morphology do not show reactivity with thi...