By Sharad Kumar, Loretta Dorstyn (auth.), Peter Erhardt, Ambrus Toth (eds.)
The skill to notice and quantify apoptosis, to appreciate its biochemistry, and to spot its regulatory genes and proteins is essential to biomedical study. In Apoptosis: tools and Protocols, moment Edition, specialist researchers describe the strategies to most sensible examine the serious steps taken with the apoptotic technique. offered from a number of diversified learn views, the quantity comprises sections protecting detection of apoptosis, detection of non-apoptotic mobile dying, adjustments of apoptotic proteins in the course of apoptosis, the research of its significant regulators, in addition to research of apoptosis in numerous organs and in version organisms. Written within the hugely profitable Methods in Molecular Biology™ sequence layout, chapters contain introductions to their respective issues, lists of the required fabrics and reagents, step by step, conveniently reproducible protocols, and notes on troubleshooting and heading off recognized pitfalls.
Comprehensive and state-of-the-art, Apoptosis: tools and Protocols, moment Edition constitutes a key technical connection with the numerous methodologies utilized in the sector, and gives rookies and skilled researchers robust instruments to light up the phenomena of programmed mobile death.
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Extra resources for Apoptosis: Methods and Protocols, Second Edition
Cell Death Differ. 14, 66–72. Ekert, P. , and Vaux, D. L. (1999). Caspase inhibitors. Cell Death Differ. 6, 1081–1086. , and Lazebnik, Y. (1997). Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells. EMBO J. 16, 2271–2281. , McStay, G. , Boucher, L. , Beere, H. , and Green, D. R. (2006). In situ trapping of activated initiator caspases reveals a role for caspase-2 in heat shockinduced apoptosis. Nat. Cell Biol. 8, 72–77. , Peterson, E. , Rasper, D. , Vaillancourt, J.
4. Assessment of Fractional DNA Content (sub-G1 fraction) 1. Cell suspension (5 × 105–1 × 106 cells/mL). 2. Cold 70% EtOH (store at −20°C). 3. 1× PBS. Flow Cytometry-Based Apoptosis Detection 23 4. 5 mL Eppendorf tubes. 5. 12 × 75 mm Falcon FACS tubes (BD Biosciences). 6. 1 mg/mL propidium iodide (PI) stock solution in PBS. Store protected from light at +4°C. Reagent is stable for over 12 months. Caution: PI is a DNA binding molecule and thus can be considered as a potential carcinogen. Always handle with care and use protective gloves.
Collect cell suspension into 12 × 75 mm Falcon FACS tube and centrifuge for 5 min, 160 × g at room temperature (RT). 2. Resuspend cell pellet in 1–2 mL of PBS and centrifuge for 5 min, 160 × g. 3. Discard supernatant and add 100 mL of TMRM staining mix. 4. Gently agitate to resuspend cell pellet. 5. Incubate for 20 min at +37°C, protected from direct light. 6. Add 500-mL PBS and keep samples on ice. 7. Analyze on a flow cytometer. Use 488-nm excitation line (Argon-ion laser or solid-state laser) and emission collected at 575 nm.