Cell Membrane Cell membrane integrity vital for proper cell functioning. Most cells can only withstand transient ruptures of the cell membrane. If the cell membrane suffers massive breaches, the cel loses the ability to accumulate and contain vital components and wil be subject to toxins from outside the cell. There is a term for cells that suffer massive cell membrane trauma - Dead Cel s.
Membrane Exclusion Trypan Blue has been used for years to assess cel viability. Trypan Blue is negatively charged, and does not bind to the cell unless the membrane is compromised. Viable cel s exclude Trypan Blue. Time sensitive. Another method to assess cell viability is dye exclusion, using an impermeant dye. If the cell takes up the impermeant dye, it is considered dead. Cel s that become reproductively non viable (ie,ionizing rad): impermeant dye is useless.
Impermeant Dyes Propidium Iodide Excited by 488 nm laser light. Emits at 620 nm (Looonnnggg emission curve). Binds to DNA, intercalating between bases with no specificity, one dye molecule/4-5 bp. 7-Aminoactinomycin D (7-AAD) Excited by 488 nm laser light. Emits at 647 nm. Binds to DNA and inserts between Cytosine and Guanine bases of double stranded DNA when the interior of the cell and chromatin is accessible. Positively stained cells are considered non-viable as 7-AAD has crossed a no longer intact cell membrane.
Apoptosis Programmed Cell Death Characterized by DNA fragmentation and in cel morphology and volume. Requires biochemical energy. Important – For the normal functioning of the immune system, embryonic development, normal tissue maintenance and chemical- and hormone-induced cell death. ‘Programmed’-the genetically determined eradication of cel s. Part of normal cell development, aging, and security mechanism. Necessary and Pathological. Removal of specific cells w/o inflammation.
Apoptosis vs. Necrosis Necrosis Toxicity-induced cell death. Requires no energy, passive. Cells swell and then karyolysis (dissolution of the chromatin and nucleus - DNase). Release of cellular contents may cause inflammation. Apoptosis ‘Stimulation’-induced cell death. Energy required. Cell shrinkage, then pyknosis (chromatin condenses), followed by karyorrhexis (fragmentation of the nucleus). Do not release cellular contents and are readily phagocytosed by macrophages.
Apoptosis vs. Necrosis From: Andreas Gewies, Intro to Apoptosis
Apoptosis Apoptotic Effects - Cell Morphology Cells change shape and shrink during apoptosis. The chromatin condenses in a process called Pyknosis. The cells become smaller and the cytoplasm shrinks around the organelles. Figure from the Cell Migration Lab, University of Reading http://www.reading.ac.uk/c ellmigration/apoptosis.htm
Apoptosis Apoptotic Effects - Cell Morphology No Treatment Treatment Cells became smaller and more granular (Blue Cells).
Apoptosis From: Wlodkowic, D (2010). Cytometry in Cell Necrobiology Revisited. Recent Advances and New Vistas. Cytometry 77A: 591-606.
Apoptosis Pathways Extrinsic (‘Death Receptor’) Intrinsic (Mitochondria) Perforin/Granzyme (T-cell mediated) From: Elmore, S. A. (2007). Apoptosis: a review of programmed cell death. Toxicol Pathol 35 (4):495-516.
Apoptosis Extrinsic (‘Death Receptor’) Ligands/Receptors implicated in the induction of apoptosis: FasL/FasR TNF- /TNFR1 Apo3L/DR3 Apo2L/DR4 Apo2L/DR5 Binding creates a death-inducing signaling complex (DISC) to be formed. Activation of procaspase-8, starts execution phase of apoptosis.
Apoptosis Intrinsic (Mitochondria) Negative stimuli - Stop the suppression of apoptosis Lack of certain growth factors, hormones or cytokines. Positive stimuli – Induce apoptosis Free radicals, radiation, hypoxia, infections, and toxins. Initiated at the inner mitochondrial membrane. Loss of mitochondrial membrane potential and the release of pro-apoptotic proteins. One set of proteins activate the caspase-dependent mitochondrial pathway. The later group activates AIF (Apoptosis-Inducing Factor) and endonuclease proteins. Bcl-2 family of proteins mediate mitochondrial apoptosis.
Apoptosis Execution Pathway Final Pathway for Apoptosis. Caspases activate cytoplasmic endonuclease and proteases. From: Elmore, S. A. (2007). Apoptosis: a review of programmed cell death. Toxicol Pathol 35 (4):495-516.
Apoptosis Apoptosis Inducing Compounds: Compound Mechanism(s) of Action Actinomycin D Caspase-3 activity increased more than 20-fold, inhibition of rRNA synthesis and the defective pre-mRNA maturation Brefeldin A caspase activation Camptothecin prevents DNA re-ligation and therefore causes DNA damage Colchicine mediated through cytochrome C release and caspase-3 activation Doxorubicin.HCl early activation of p53 in tumor cel s that was fol owed by caspase-3 activation and DNA fragmentation Ionomycin induces the activation of calcium-dependent endonuclease Mitomycin C Inhibitor of DNA synthesis Staurosporine caspase-independent and dependent mechanisms Thapsigargin Bax-dependent signaling pathway control ing the cytosolic release of mitochondrial apoptogenic molecules
Apoptosis Assays Apoptosis is a complex and tightly regulated phenomenon. Many assays are available. Advantages and Disadvantages: Apoptosis and Necrosis have overlapping phenomena. Do you want to detect initiation of apoptosis? Or the execution phase? Kinetics of cell death can be tricky. Caspases may be expressed only transiently, you may miss your time window because apoptotic cells may die quickly, and perhaps only a few cells are apoptotic at the time of analysis.
Apoptosis Assays Where in the pathway? DNA Fragmentation -TUNEL assay. Caspase Detection – PhiPhiLux and Vybrant FAM Poly Caspases Assay Kit. Chromatin Condensation - Vybrant® DyeCycle™ Violet/SYTOX® AADvanced™ Apoptosis Kit for Chromatin Condensation (Invitrogen cat # A35135). Membrane Alteration – Annexin V, and Membrane Permeability. Mitochondria - MitoProbe™ JC-1 Assay Kit for Flow Cytometry (Invitrogen cat # M34152).
TUNEL Assay TUNEL (Terminal dUTP Nick-End Labeling) During Apoptosis, Genomic DNA is cleaved into small double- stranded fragments and single-stranded breaks called ‘nicks’. Terminal deoxynucleotidyl transferase (TdT) labels DNA strand breaks by catalyzing the polymerization of labeled nucleotides to free 3’-OH DNA ends. The 3′-OH ends of the breaks can be detected by attaching a fluorochrome. This is generally done directly or indirectly (biotin) using fluorochrome-labeled deoxynucleotides in a reaction catalyzed preferably by TdT. Best results are achieved using a positive control (fixed, permeabilized cells treated with DNase) and a negative control (no FITC labeling reagent). We have had good luck with the Roche kit (cat # 11 684 795 910).
TUNEL Assay Gated on Sperm Negative Control Tunel FITC, No PI Positive Control No Tunel, PI Only Positive Control Tunel FITC and PI Positive Control Tunel FITC and PI Test
TUNEL Assay Sperm Tunel Assay 2/29/2012 Al values are percentages. TUNEL FITC Test TUNEL FITC Test TUNEL FITC Test TUNEL FITC Test Standard Date Patient Negative Control Positive Control Rep. 1 Rep. 2 Rep. 3 Rep. 4 Mean Deviation 11/18/2011 #111 1.4 93 19.1 19 18.6
28.33 6.33 Excluded from mean and standard deviation. <50%
TUNEL Assay Advantages Assay is very sensitive. Can detect ~100 cells (flow cytometry). Fast, can be completed in 3 hours. High reproducibility, with good precision. Paraformaldehyde fixation before permeabilization prevents the loss of small fragments of DNA. Disadvantages We do not know how many strand breaks are necessary for detection. Necrotic cells can generate false positives. Detergent is used to permeabilize the cells. Apoptotic cells in saline + detergent are extremely fragile and can be lysed when pipetted or vortexed.
Caspase Assay PhiPhiLux staining from OncoImmunin, Inc. A fluorescent cmpd is dimerized w/ a peptide linker. The proximity of the 2 cmpds quenches their fluorescence. Cmpd is taken up by cells. Linker (DEVDGI) is specific for Caspase 3. When the linker is cut they fluoresce. Available in green and red substrates. Add PI, PI+ cells are not green as the PhiPhiLux reagent diffuses out.
Caspase Assay PhiPhiLux staining from OncoImmunin, Inc. From: Packard, BZ. (2008). Intracellular protease activation in apoptosis. Cell Research 18: 238-247.
Caspase Assay Advantages PhiPhiLux reagents Caspase 1, 6, 8 and 9 Available at green and red emission spectra. Invitrogen Caspase kits. Disadvantages Kinetics – When are the caspases active in apoptosis? Need to run with a potent inducer of apoptosis.
Chromatin Condensation Assay Chromatin Condensation/Dead Cell Apoptosis kit. 2 Stains. Vybrant DyeCycle Violet stains condensed chromatin more brightly than normal cell chromatin. SYTOX AADvanced stain only stains necrotic cells with permeable membranes.
Annexin V Assay Timeline 1990 Andree at al. found that a protein, Vascular Anticoagulant , bound to phospholipid bilayers in a calcium dependent manner. Protein was renamed Annexin V. 1992 Fadok et al. discovered that macrophages specifically recognize phospatidylserine (PS) that is exposed on the surface of lymphocytes during the development of apoptosis. This PS is normally on the inner leaflet of the membrane. 1994 Koopman et al. developed a flow cytometric assay for measuring FITC conjugated Annexin V binding to apoptotic cells. Stained control and serum starved cells with ethidium bromide and Annexin V-FITC.
Annexin V Assay Normal Cell Membrane Apoptotic Cell Membrane Apoptotic/Necrotic Cell No PS on surface. PS on surface. Membrane PS on surface, membrane disintegrates. Inner Leaflet Inner Leaflet Inner Leaflet = Phosphatidylserine
Annexin V Assay Figure from Imgenex, CytoGLO Annexin V FITC kit
Annexin V Assay Titrate the Annexin V.
Annexin V Assay Must perform compensation. Annexin V-FITC vs. PI, negligible with 552 nm Green laser. Annexin V can be conjugated to many other fluorochromes. Compensation. Your PI and Annexin V-FITC controls - apoptotic or necrotic for proper compensation. If not, no Annexin V-FITC binding and PI cannot cross a healthy cel membrane. Induce cel injury.
Annexin V Assay Unstained Cells, no treatment, live cells Data kindly provided by Patrick Grogan, Cohen Lab
Annexin V Assay Propidium Iodide Only, cells – not healthy These cells were treated with withaferin A.
Annexin V Assay Annexin-FITC Only, cells – not healthy 4.5% These cells were treated with withaferin A. Your PI and Annexin V cells must be apoptotic or necrotic for proper compensation.
Annexin V Assay Untreated, Annexin + PI
Annexin V Assay 2 uM WA Rx, Annexin + PI 8.4% 16.8%
Annexin V Assay 6 uM WA Rx, Annexin + PI 5.8% 57.3% 30.1% 6.8%
Annexin V Assay Concerns Very important to have negative and positive controls for live and apoptotic cells. Trypsinization can lead to Annexin-binding. False positives. Annexin V only binds in the presence of divalent cations (Ca2+ and Mg2+). Removal of the cations results in rapid dissociation of PS and Annexin. An overly long incubation period - nonspecific binding.
MitoProbe JC-1 Assay Mitochondria in Apoptosis Intracellular energy – produced by mitochondrial respiratory chain. Stored as an electrochemical gradient. Trans-membrane electrical ~180 to 200 mV. Negative charge (inside). The membrane potential ( ) of mitochondria drives the production of ATP. A possible early apoptotic event is the collapse of the , or later after the loss of DNA integrity.
MitoProbe JC-1 Assay JC-1 Lipophilic cation. Excited by 488 nm laser. Fluorescence emission changes based on and is reversible. The dye forms aggregates that emit at ~590 nm, while the monomeric form emits at ~530 nm. Aggregate formation starts at 80-100 mV and peaks at ~200 mV. Red fluorescence means happy mitochondria, green fluorescence means a drop in . Qualitative – shift from green to red. Quantitative – measure absolute values of green and red emission.
MitoProbe JC-1 Assay Treat cel s with Fccp. Fccp depolarizes the mitochondrial membrane potential and induces apoptosis.
MitoProbe JC-1 Assay Concerns The may be affected by a multitude of factors. Apoptosis can be triggered by many different stimuli, effecting many intracellular systems. Apoptotic DNA fragmentation may occur before a change in mitochondrial , in this instance, the change in is because of apoptosis, not a trigger of apoptosis. We can stain cells for extracellular markers, as wel as JC-1, but care must be taken to compensate properly.
Andree, H. A. M. et al. (1990) Binding of Vascular Anticoagulant (VAC ) to Planar Phospholipid Bilayers. The Journal of Biological Chemistry 265, 9, 4923- 4928.
Cassarizza, A et al. (2000) Flow Cytometric Analysis of Mitochondrial Membrane Potential using JC-1. Current Protocols in Cytometry 9.14, 1-7.
Elmore, S. A. (2007). Apoptosis: a Review of Programmed Cell Death. Toxicol Pathol 35 (4):495-516.
Janeway’s Immunology, Seventh Edition, 2008.
Fadok, V. et al. (1992) Exposure of Phosphatidylserine on the Surface of Apoptotic Lymphocytes triggers specific recognition and removal by Macrophages. The Journal of Immunology 148, 2207-2216.
Koopman, G. et al. (1994) Annexin V for Flow Cytometric Detection of Phosphatidylserine Expression on B cells undergoing Apoptosis. Blood 84, 1415-1420.
Lugli, E. et al. (2007) Polychromatic Analysis of Mitochondrial Membrane Potential using JC-1. Current Protocols in Cytometry 7.32, 1-15.
Packard, B. et al. (2008) Intracellular Protease Activation in Apoptosis and Cell- mediated Cytotoxicity Characterized by Cell-permeable fluorogenic protease substrates. Cell Research 18, 238-247.
Pozarowski, P. et al. (2003) Flow cytometry of Apoptosis. Current Protocols in Cytometry, 7.19, 1-33.
Telford, W. et al. (2004) Chapter 8: Multiparametric analysis of apoptosis by flow and image cytometry. Methods in Molecular Biology: Flow Cytometry Protocols, 2nd ed. 141-159.
Wlodkowic, D. et al. (2010). Cytometry in Cell Necrobiology Revisited. Recent Advances and New Vistas. Cytometry 77A: 591-606.