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Detection of Reactive Oxygen Species Using Mito. SOX and Cell. ROX in Zebrafish BIO PROTOCOLAbstract. The production of free radicals is the result of normal cellular metabolism. Free radicals are involved in innumerable different cellular and biological functions such as signaling, proliferation, cell death, aging, inflammation, etc. Under physiological conditions, the levels of reactive oxygen species ROS are strictly regulated by the cells. However, during stressful conditions such as oxidative stress, ROS levels increase causing damages to different molecules like DNA, lipids and proteins. Increased levels of ROS have been associated with a growing list of different diseases. In this protocol, we used Mito. SOX and Cell. ROX Green oxidative stress probes to label the intracellular ROS and detect the fluorescence using cell sorting and confocal analyses. Keywords Zebrafish, Reactive Oxygen Species, Oxidative stress. Background Dysfunction of regulatory T Treg cells has been detected in diverse inflammatory disorders, including chronic graftversushost disease GVHD. Issuu is a digital publishing platform that makes it simple to publish magazines, catalogs, newspapers, books, and more online. Easily share your publications and get. The production of free radicals is the result of normal cellular metabolism. Free radicals are involved in innumerable different cellular and biological functions. Diva7FcsExportOptions.png' alt='Download Bd Facsdiva Software Free' title='Download Bd Facsdiva Software Free' />
In biotechnology, flow cytometry is a laser or impedancebased, biophysical technology employed in cell counting, cell sorting, biomarker detection and protein. Original Article. Inducible Apoptosis as a Safety Switch for Adoptive Cell Therapy. Antonio Di Stasi, M. D., SiokKeen Tey, M. D., Gianpietro Dotti, M. D., Yuriko Fujita. Gua de tubos BD Vacutainer para recoleccin venosa. Tapn Tapn Hemogard convencional. Aditivo. Mezclado. Activador de coagulacin y gel. Materials and Reagents. Petri dishes Thermo Fisher Scientific, Fisher Scientific, catalog number FB0. Z. 1. 5 ml tubes Eppendorf, catalog number 0. TC Treated culture dish Corning, catalog number 4. Falcon round bottom polystyrene 5 ml tubes Corning, Falcon, catalog number 3. Sterile 4. 0 m cell strainer Corning, catalog number 4. Cellvis, catalog number D3. N. 3 ml transfer pipette Sigma Aldrich, catalog number Z1. Zebrafish adults wild type or mutant strains. Sodium chloride Na. Cl Sigma Aldrich, catalog number S7. Potassium chloride KCl Sigma Aldrich, catalog number P9. Calcium chloride dihydrate Ca. Cl. 22. H2. O Sigma Aldrich, catalog number C5. Magnesium sulfate heptahydrate Mg. SO47. H2. O Sigma Aldrich, catalog number 6. H2. O Sigma Aldrich, catalog number W4. DMEMF 1. 2 medium Thermo Fisher Scientific, Gibco. TM, catalog number 1. Fetal bovine serum FBS Thermo Fisher Scientific, Gibco. TM, catalog number 1. Tricaine Sigma Aldrich, catalog number A5. EDTA 0. 5, no phenol red Thermo Fisher Scientific, Gibco. TM, catalog number 1. Tris EDTA buffer solution p. H 9. 0 Sigma Aldrich, catalog number SRE0. Collagenase P Sigma Aldrich, catalog number COLLP RO. Phosphate buffered saline PBS, p. H 7. 4 Thermo Fisher Scientific, Gibco. TM, catalog number 1. Hanks balanced salt solution HBSS, with calcium and magnesium, no phenol red HBSSCaMg Thermo Fisher Scientific, Gibco. TM, catalog number 1. Mito. SOXTM red mitochondrial superoxide indicator Thermo Fisher Scientific, Molecular Probes. TM, catalog number M3. Dimethyl sulfoxide DMSO Sigma Aldrich, catalog number D2. D 7 AAD BD, BD Pharmingen. TM, catalog number 5. Annexin V APC BD, BD Pharmingen. TM, catalog number 5. Annexin V binding buffer, 1. BD, BD Pharmingen. TM, catalog number 5. Cell. ROX green reagents, for oxidative stress detection Thermo Fisher Scientific, Molecular Probes. TM, catalog number C1. Cell. ROX green flow cytometry assay kit Thermo Fisher Scientific, Molecular Probes. TM, catalog number C1. Certified. TM low melting agarose Bio Rad Laboratories, catalog number 1. Instant Ocean aquarium sea salt mixture Spectrum Brand, Instant Ocean, catalog number SS1. Recipes. DMEMF 1. Recipes. 6. 0x E3 embryo medium stock solution see Recipes. Homogenization medium see Recipes. M Mito. SOX stock solution see Recipes. Dissecting forceps microdissection DR Instruments, catalog number 1. Ultrafine single deer hair with handle Ted Pella, catalog number 1. Thermoblock Sigma Aldrich, catalog number Z6. Manual pipette PR 2. Mettler Toledo, Rainin, catalog number 1. Standard tabletop centrifuges Eppendorf, catalog number 0. Dissecting microscope Leica, model Leica S8 APO. FACS Fluorescence activated cell sorter e. FACSAria II equipped with FACSDiva software using the blue 4. BD. Confocal microscope e. LSM 5. 10 NLO Meta system mounted on an Axiovert 2. M microscope Carl Zeiss with a Plan Apochromat 1. Plan Apochromat 2. FACSDiva software. Single cell homogenization and ROS Mito. SOX and Cell. ROX Green staining. Grow zebrafish embryos at 2. C in 2. 5 ml of fish water or 1x E3 embryo medium until the desired stage. Pre heat the homogenization solution and the DMEM 1. Advanced Email Extractor Pro Registration Cracked Screen. FBS at 2. 8 C. Collect the embryos and manually dechorionate them with a pair of forceps with sharp tips after 2. Use one forceps to hold the external membrane chorion, then make a tear using the other forceps and gently remove the chorion. Transfer dechorionated embryos to 1. Note The quality of the homogenization will depend on the stage and the number of embryos used. Do not exceed the number of 5. Remove most of the water or 1x E3 embryo medium without damaging or drying the embryos. Euthanize embryos with 4. Wash twice with 1 ml of 1x PBS for 1 2 min. Add 6. 00 l of homogenization solution collagenase P trypsin EDTA in 1x PBS and incubate at 2. C in a thermoblock for 2. During the incubation time, periodically every 1 2 min homogenate the samples pipetting up and down with a 2. During the incubation time a progressive homogenization of the samples should be noted. Add 7. 00 l of DMEM 1. FBS, mix by pipetting or vortexing and then centrifuge the cells for 2 min at 6. RT. Discard supernatant and resuspend the cells in 1 ml 1x PBS. Centrifuge the cells for 2 min at 6. Resuspend the cells in 1 ml of DMEM 1. FBS. Keep the cells at 2. C in DMEM 1. 0 FBS until the staining in order to increase the survival of the cells. Note Alternatively, the cells can be stored at 4 C in order to minimize the level of oxidative stress induced by cell homogenization. However, we noticed an increased level of apoptotic and dead cells with the incubation at 4 C. Aliquots the cells in different tubes and stain the cells with the different staining solutions at 2. C in a thermoblock in the dark. Oxidative stress Mito. SOX and Cell. ROX Green, apoptosis and cell death staining. Centrifuge the cells for 2 min at 6. RT. Remove supernatant and resuspend the cells in prewarmed HBSSCaMg at 2. C. Centrifuge the cells, remove the supernatant and stain the cells with 4. M solution of Mito. SOX in HBSSCaMg for 1. M solution of Cell. ROX Green in DMEM 1. FBS for 3. 0 min at 2. C, in the dark. Wash the cells three times with 1 ml of prewarmed HBSSCaMg to remove the excess of the probes. To test the viability of the cells after homogenization, an Annexin V APC and 7 AAD double staining can be performed in parallel for the oxidative stress staining. For Annexin V APC and 7 AAD double staining cells were centrifuged, washed with 5. C from Annexin V APC kit and then incubated in 6. C in the dark. After the staining, wash the cells three times with 5. Filter the cell solution through a sterile 4. FACS analysis. Transfer the cells to a 5 ml tube and then sort the cells with a FACS. Flow cytometry data were collected on a FACSAria II equipped with FACSDiva software using the blue 4. AAD 5. 506. 47 nm, ExcitationEmission peaks, Cell. ROX green Cell. ROX green flow cytometry assay kit 5. Cell. ROX green reagent 4. Mito. SOX 5. 105. BD for APC signal 6. Figure 2 for representative analysis. Whole mount staining of ROS Mito. SOX and Cell. ROX Green. Grow zebrafish embryos at 2. C in 2. 5 ml of fish water or 1x E3 embryo medium until the desired stage. Collect the embryos and manually dechorionate them with forceps, if necessary. Transfer the embryos to a 1.