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Solutions to Detect Apoptosis and Pyroptosis Powerful Tools for In vitro Fluorescent Imaging in Cancer Research Jackie Carville May 11, 2016 About Us Products and Services • Custom Assay Services – Immunoassay design and development – Antibody purification/conjugation • Consumable Reagents – ELISA Solutions – Cell-permeant Fluorescent Probes for detection of cell death • Products for research use only. Not for use in diagnostic procedures. ELISA Solutions • ELISA Wash Buffer • PBS • Substrates • Stop Solutions • Plates • Coating Buffers • Blocking Buffers • Conjugate Stabilizers • Assay Diluents • Sample Diluents Custom Services • Antibody Conjugation • Immunoassay Design • Immunoassay Development • Lyophilization • Plate Coating Overview of ICT’s Fluorescent Probes FOR DETECTION OF: • Active caspases • Caspases/Cathepsins in real time • Mitochondrial Health • Cytotoxicity • Serine Proteases Today’s Agenda • FLICA • FLISP • Magic Red • Cell Viability • Mito PT • Oxidative Stress • Cytotoxicity FLICA • FLICA – Fluorescent Labeled Inhibitor of Caspases • In vitro, whole cell detection of caspase activity in apoptotic or caspase-positive cells • Available in green, red, and far red assays FLICA Cancer Research Applications • FLICA is the most accurate and sensitive method of apoptosis detection • Can identify four stages of cell death in one sample • Detect poly caspase activity or individual caspase enzymes How FLICA A. INACTIVE CASPASE (ZYMOGEN) works… prodomain B. CASPASE PROCESSING FMK Fluorescent Reporter YVAD tag C. ACTIVATED CASPASE (HETERO-TETRAMER) D. BINDING OF FLICA Sample flow results… 660 FLICA Poly Caspase (VAD): FAM FLICA Caspase-3/7 (DEVD): 660 FLICA Caspase-1 (YVAD): Jurkat cells treated with Jurkat cells treated with THP-1 cells (undifferentiated) staurosporine staurosporine stimulated with LPS and ATP (Kit # 9120) (Kit # 94) for 24 hours (Kit # 9122) Red – Control (non induced) Black – Experimental (apoptotic) Sample plate reader results… Poly Caspase Detection (SR- • Jurkat cells were VAD-FMK, Cat # 917) treated with 60.0 staurosporin for 4 50.0 40.0 hrs, then washed RFU and read using 30.0 fluorescence plate 20.0 reader (Ex/Em = 10.0 550/590) 0.0 Non-Induced Induced Sample fluorescent microscopy results… THP-1 cells dually stained with FAM- Salmonella Infected epithelial cells FLICA Poly Caspase Assay and Hoechst labeled with FAM-FLICA Caspase-1 (Kit # 92) Assay (image courtesy of Knodler, et al. 2010. PNAS 107 (41) 17733-8) (Kit # 98) Magic Red • Cell-permeant fluorescent substrate probes used to quantitate caspase or cathepsin activity in cultured cells • Monitor caspase and cathepsin activity in real time • Available for cathepsin B, K, or L, as well as caspases 3&7 Magic Red Cancer Research Applications • Upregulated cathepsin B and L – Cancer of the colon, pancreas, ovaries, breast, lung, and skin • Upregulated cathepsin K – Lung tumors – Degenerative bone disease How Magic Red Works… • The target substrate peptide sequence is linked to a red fluorophore, Magic Red • Magic Red fluoresces red when cleaved by the specific enzyme • As protease activity increases, more substrate is cleaved, and red fluorescent signal increases • Watch color develop over time – analyze with fluorescent microscope or plate reader Sample Magic Red Results Rat fibroblasts were seeded in a 12-well plate at 1x104 cells/mL and irradiated the following day. 23.1 µL of MR-(DEVD)2 solution was mixed with media and added. 1 hour later, 300 µL media was added and cells were photographed over 16 hours. Bright field images show increasing red intensity as caspase activity and apoptosis progressed (Dr. Martin Purschke, Massachusetts General Hospital). Sample Magic Red Results Cathepsin B-positive THP-1 cells fluoresce red after staining with MR-(RR)2. Red fluorophore concentrates inside lysosomes. Sample Magic Red Results Cells were dually stained using ICT’s Magic Red® substrate and Hoechst 33342 nuclear stain. Positive cells bearing orange-red lysosomal bodies with less intense blue nuclei are intermixed with negative cells with absent or reduced orange-red lysosomal staining and bright blue nuclei. Photo provided by Dr. Zbigniew Darzynkiewicz at Brander Cancer Research Center Institute, New York City, NY MitoPT • Quantitate mitochondrial functionality and apoptosis • Identify a collapse in the electrochemical gradient across the mitochondrial membrane, as measured by the change in the membrane potential • Available in JC-1, TMRE, and TMRM kits MitoPT Cancer Research Applications • Mitochondria play a central role in the biochemical processes associated with the life and death stages of eukaryotic cells • Also tied to neurodegenerative diseases How MitoPT Works (JC-1) • Lipophilic JC-1 dye, bearing a delocalized positive charge, enters the negatively charged mitochondria where it accumulates • When the mitochondrial ΔΨm collapses in apoptotic cells, MitoPT JC-1 no longer accumulates inside the mitochondria, instead becoming more evenly distributed throughout the cytosol • Healthy cells fluoresce orange and green • Cells with compromised mitochondria fluoresce green and exhibit lower levels of orange fluorescence Sample MitoPT Results Normal healthy cells (two cells, upper and lower left), containing mitochondria with polarized inner membranes, concentrate MitoPT® JC-1 and fluoresce bright orange. Apoptotic cells (three cells, right), bearing mitochondria of various stages of permeability, exhibit a reduced orange fluorescence relative to the healthy cell population and increased green fluorescence, as the reagent becomes dispersed throughout the cells. How MitoPT Works (TMRM and TMRE) • Lipophilic TMRE or TMRM dye, bearing a delocalized positive charge, enters the negatively charged mitochondria where it accumulates and fluoresces orange upon excitation • When the mitochondrial ΔΨm collapses in apoptotic cells, MitoPT TMRE or TMRM no longer accumulate inside the mitochondria, instead becoming more evenly distributed throughout the cytosol • Healthy cells fluoresce orange • Cells with depolarized mitochondria exhibit lower levels of orange fluorescence Sample MitoPT Results Cytotoxicity • Basic – Measure necrotic and membrane-compromised cells in a mixed- population sample of effector and target cells. • Total – Further distinguish apoptosis from necrosis in target cells, leading to a more accurate assessment of cytolytic activity. • Does not use 51CR Cytotoxicity Cancer Research Applications • Cytolytic activity is an important process for eliminating intracellular pathogens and cancer cells • Accomplished through natural killer (NK) leukocytes • Activity of NK cells, and their effect on target cells, is frequently studied in immunomodulation How Our Basic Cytotoxicity Tests Work… How our Total Cytotoxicity Tests Work… Sample Cytotoxicity Test Results Basic Total FLISP • Fluorescent Labeled Inhibitors of Serine Proteases • Assess the intracellular levels of chymotrypsin-like serine protease activity in vitro FLISP Cancer Research Applications • Activated serine proteases play major roles in: – Apoptosis – Markers of tumor malignancy – Diagnostic and prognostic indicators of breast carcinomas – Neck and head carcinomas • Activity is also altered in a variety of other cell-mediated diseases related to transplant rejection and infections How FLISP Works… • The target inhibitor peptide sequence is linked to a red, SR-101, or a green fluorophore, FAM • FFCK kit to detect chymotrypsin-like serine proteases that favor phenylalanine • FLCK kit for those that favor leucine • Use FLISP in combination with FLICA to discriminate serine protease activity from caspase activity in the same cell Sample FLISP Results Cells with active serine proteases stain green with FFCK along the Y-axis and cells with active caspases stain red with SR-VAD-FMK along the X-axis. Colocalization of serine protease activity versus caspase activity is evident in dually stained cells (B, C, D, and G). Activation of caspases was rapidly followed by serine protease activation and the time between was relatively short as very few cells are just red (E). Data courtesy of Dr. Jerzy Grabarek, Brander Cancer Center, NY, and Pomeranian School of Medicine, Szczecin, Poland. Cell Viability • Necrosis vs. Apoptosis Assay Kit • “How healthy is my cell culture?” webinar Oxidative Stress • Glutathione Assay • Hydrogen Peroxide Assay • DNA Damage ELISA Assay • Nitric Oxide • Coming soon: Total ROS • Webinar in June! Resources • Webinars • Videos • Blog • Technical documents Excellent Customer and Technical Support Thank You Jackie Carville Marketing Director [email protected] 1-800-829-3194 www.immunochemistry.com.
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