Sytox red dead cell stain, by Thermo Fisher Scientific - Product details

1

Dissociation and Sorting of Tumor Cells

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Tumors were dissected, minced, and digested rotating for 30 min at 37°C with 1 mg/mL Collagenase I (Worthington Biochemical LS004194), 3 mg/mL Dispase II (Roche 04942078001), and 0.1 mg/mL DNase I (Sigma D4527) in PBS. Following digestion, cells were incubated with EDTA to 10 mM at room temperature for 5 min. Cells were then filtered through a 70 μm strainer and washed twice with PBS. Single cell suspensions were resuspended in flow cytometry staining buffer (Thermo Fisher 00-4222-57) and first stained with 10 μL of CD16/CD32 monoclonal antibody (Thermo Fisher 14-0161-82) for 15 min to block Fc receptors and then stained using with antibodies to CD45-APC-Cy7 (BD 557659) at 1:100 dilution followed by SYTOX Red Dead Cell Stain (Life Technologies S34859) at 1:1000 dilution to visualize dead cells. All antibodies were incubated for 15–20 min on ice and then washed. Cell sorting was performed with a BD FACS Aria and data was analyzed with FlowJo Software (BD).

Lau A.N., Li Z., Danai L.V., Westermark A.M., Darnell A.M., Ferreira R., Gocheva V., Sivanand S., Lien E.C., Sapp K.M., Mayers J.R., Biffi G., Chin C.R., Davidson S.M., Tuveson D.A., Jacks T., Matheson N.J., Yilmaz O, & Vander Heiden M.G. (2020). Dissecting cell-type-specific metabolism in pancreatic ductal adenocarcinoma. eLife, 9, e56782.

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2

Fluorescence-Activated Cell Sorting of Zebrafish HSPC

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FACS analysis was performed using Tg(flk1:dsRed;cmyb:GFP) or Tg(−6.0itga2b:egfp) HSPC reporter embryos as previously described. Briefly, embryos (5 pooled embryo per sample × ≥3 replicates) were dissociated, resuspended in 1×PBS, and analyzed by BD FACSCanto II (BD Biosciences, San Jose CA) in the presence of SYTOX Red Dead Cell Stain (5nM; Life Technologies, Waltham MA). Data was analyzed using FlowJo X software (TreeStar, Ashland OR), and expressed as ratio of control. For Tg(−6.0itga2b:egfp) embryos, CD41^lo cells were gated. For isolation of endothelial (Flk1:dsRed⁺cMyb:GFP⁻) and HSPC (Flk1:dsRed⁺cMyb:GFP⁺) fractions, pooled embryos (n>1000) were sorted at 48hpf by FACSAria (BD Biosciences, San Jose CA) with RNA isolation as above.

Kwan W., Cortes M., Frost I., Esain V., Theodore L.N., Liu S.Y., Budrow N., Goessling W, & North T.E. (2016). Central Nervous System Regulation of Embryonic HSPC Production via Stress-Responsive Glucocorticoid Receptor Signaling. Cell stem cell, 19(3), 370-382.

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3

Quantifying Cellular Uptake and Localization

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GFP-22 siRNA was purchased from Qiagen (USA) and scrambled siRNA (custom 22 mer) was purchased from GE Healthcare Dharmacon (USA) (Sense Strand: 5’- GCA AGC TGA CCC TGA AGT TC (dTdT)-3’, anti-sense strand 3’- GAA CUU CAG GGU CAG CUU GC-5’, Mol. Wt. 13,965.6 (g/mol), the oligonucleotide has been converted to the 2’-hydroxyl, and annealed), Inc. MTS reagent was purchased from Promega (USA). Lipofectamine^® 2000 (L2K) transfection reagent, SYTOX^® Red dead cell stain, LysoTracker^® Blue DND-22, and AlexaFluor^®680 Wheat Germ Agglutinin- (AlexaFluor^® 680 WGA) conjugate were purchased from Life Technologies (USA). Label IT^® siRNA Tracker intracellular localization kit (Cy3) was obtained from Mirus (USA). Fetal Bovine Serum (FBS), DMEM and RPMI media, sodium pyruvate, PBS and trypsin, were purchased from Atlanta Biologicals (USA). Mark-tubes made of borosilicate glass #50, (L= 80 OD= 1.50 Wall= 0.01 mm) were purchased from Hilgenberg GmbH (Germany).

Badwaik V.D., Aicart E., Mondjinou Y.A., Johnson M.A., Bowman V.D, & Thompson D.H. (2016). Structure-Property Relationship for in Vitro siRNA Delivery Performance of Cationic 2-Hydroxypropyl-β-cyclodextrin: PEG-PPG-PEG Polyrotaxane Vectors. Biomaterials, 84, 86-98.

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4

Fluorescent Embryo Cell Sorting

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Fluorescent embryos (5 embryos/sample × 4 replicates/condition) were dissociated, resuspended in × PBS, and analyzed on a BD FACS Canto II in the presence of SYTOX Red dead cell stain (5 nM, Life Technologies) as described previously (Cortes et al., 2015 (link)). Data were analyzed using FlowJo X software (Tree Star). Double-negative, Flk1⁺/cMyb⁻, and Flk1⁺/cMyb⁺ cells were sorted (1,000 embryos/sample × 3 replicate sorts) on a BD SORP FACS Aria (BIDMC Flow Cytometry Core).

Cortes M., Chen M.J., Stachura D.L., Liu S.Y., Kwan W., Wright F., Vo L.T., Theodore L.N., Esain V., Frost I.M., Schlaeger T.M., Goessling W., Daley G.Q, & North T.E. (2016). Developmental Vitamin D Availability Impacts Hematopoietic Stem Cell Production. Cell reports, 17(2), 458-468.

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5

ALDH-positive Cell Isolation and Sorting

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ALDEFLUOR kit (Stem Cell Technologies, Vancouver, Canada) was used according to the manufacturer's instructions. ALDH-positive cells were defined as the cells that displayed greater fluorescence compared with a control staining reaction containing the ALDH inhibitor, DEAB (diethylaminobenzaldehyde), upon addition of the synthetic ALDH substrate BAAA. Cell sorting was performed using a FACSAria flow cytometer (Becton Dickinson, Mississauga, Canada). Dead cells positive to SYTOX Red Dead Cell Stain (Life Technologies, Grand Island, NY) were excluded.

Canino C., Luo Y., Marcato P., Blandino G., Pass H.I, & Cioce M. (2015). A STAT3-NFkB/DDIT3/CEBPβ axis modulates ALDH1A3 expression in chemoresistant cell subpopulations. Oncotarget, 6(14), 12637-12653.

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6

Fluorescent Embryo Cell Sorting

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Fluorescent embryos (5 embryos/sample × 4 replicates/condition) were dissociated, resuspended in × PBS, and analyzed on a BD FACS Canto II in the presence of SYTOX Red dead cell stain (5 nM, Life Technologies) as described previously (Cortes et al., 2015 (link)). Data were analyzed using FlowJo X software (Tree Star). Double-negative, Flk1⁺/cMyb⁻, and Flk1⁺/cMyb⁺ cells were sorted (1,000 embryos/sample × 3 replicate sorts) on a BD SORP FACS Aria (BIDMC Flow Cytometry Core).

Cortes M., Chen M.J., Stachura D.L., Liu S.Y., Kwan W., Wright F., Vo L.T., Theodore L.N., Esain V., Frost I.M., Schlaeger T.M., Goessling W., Daley G.Q, & North T.E. (2016). Developmental Vitamin D Availability Impacts Hematopoietic Stem Cell Production. Cell reports, 17(2), 458-468.

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7

Multiparametric Flow Cytometry of Tumor Microenvironment

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For flow cytometric analysis of in vivo experiments, blood, spleen, and tumor were harvested at either day 16 or 18 post tumor implantation. Single cell suspensions were prepared and red blood cells were lysed using ACK Lysis Buffer (Life Technologies). Live/dead cell discrimination was performed using Live/Dead Fixable Aqua Dead Cell Stain Kit (Life Technologies) or Sytox Red Dead Cell Stain (Life Technologies). Cell surface staining was done for 20–30 minutes. Intracellular staining was done using a fixation/permeabilization kit (00-5521-00, eBioscience.) T effector cells were phenotyped as CD8⁺CD44⁺, myeloid derived suppressor cells (MDSC) as CD11b⁺Gr-1⁺, and regulatory T cells (Tregs) as CD4⁺FOXP3⁺. All flow cytometric analysis was done using an LSR II (BD) or FACSCalibur (BD) and analyzed using FlowJo software (TreeStar) or the FlowCore package in the R language and environment for statistical computing. See Supplementary Methods for a list of antibodies used.

Victor C.T., Rech A.J., Maity A., Rengan R., Pauken K.E., Stelekati E., Benci J.L., Xu B., Dada H., Odorizzi P.M., Herati R.S., Mansfield K.D., Patsch D., Amaravadi R.K., Schuchter L.M., Ishwaran H., Mick R., Pryma D.A., Xu X., Feldman M.D., Gangadhar T.C., Hahn S.M., Wherry E.J., Vonderheide R.H, & Minn A.J. (2015). Radiation and Dual Checkpoint Blockade Activates Non-Redundant Immune Mechanisms in Cancer. Nature, 520(7547), 373-377.

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8

Multiparametric Flow Cytometry of Tumor Microenvironment

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For flow cytometric analysis of in vivo experiments, blood, spleen, and tumor were harvested at either day 16 or 18 post tumor implantation. Single cell suspensions were prepared and red blood cells were lysed using ACK Lysis Buffer (Life Technologies). Live/dead cell discrimination was performed using Live/Dead Fixable Aqua Dead Cell Stain Kit (Life Technologies) or Sytox Red Dead Cell Stain (Life Technologies). Cell surface staining was done for 20–30 minutes. Intracellular staining was done using a fixation/permeabilization kit (00-5521-00, eBioscience.) T effector cells were phenotyped as CD8⁺CD44⁺, myeloid derived suppressor cells (MDSC) as CD11b⁺Gr-1⁺, and regulatory T cells (Tregs) as CD4⁺FOXP3⁺. All flow cytometric analysis was done using an LSR II (BD) or FACSCalibur (BD) and analyzed using FlowJo software (TreeStar) or the FlowCore package in the R language and environment for statistical computing. See Supplementary Methods for a list of antibodies used.

Victor C.T., Rech A.J., Maity A., Rengan R., Pauken K.E., Stelekati E., Benci J.L., Xu B., Dada H., Odorizzi P.M., Herati R.S., Mansfield K.D., Patsch D., Amaravadi R.K., Schuchter L.M., Ishwaran H., Mick R., Pryma D.A., Xu X., Feldman M.D., Gangadhar T.C., Hahn S.M., Wherry E.J., Vonderheide R.H, & Minn A.J. (2015). Radiation and Dual Checkpoint Blockade Activates Non-Redundant Immune Mechanisms in Cancer. Nature, 520(7547), 373-377.

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9

ALDEFLUOR Assay for ALDH Detection

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The ALDEFLUOR kit (Stem Cell Technologies, Vancouver, Canada) was used according to the manufacturer's instructions. ALDH-positive cells were defined as the cells that displayed greater fluorescence compared with a control staining reaction containing the ALDH inhibitor, DEAB (diethylaminobenzaldehyde), upon addition of the synthetic ALDH substrate BAAA. In some experiments, dead cells positive to SYTOX Red Dead Cell Stain (Life Technologies, Grand Island, NY) were excluded.

Pass H.I., Lavilla C., Canino C., Goparaju C., Preiss J., Noreen S., Blandino G, & Cioce M. (2016). Inhibition of the colony-stimulating-factor-1 receptor affects the resistance of lung cancer cells to cisplatin. Oncotarget, 7(35), 56408-56421.

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10

Apoptosis Quantification by Flow Cytometry

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Cells were plated and treated in 6 well plates as described above. After 24 h of treatment, 500,000 cell aliquots of each sample were taken, including 4 aliquots of the 10 μM Etoposide positive control and 1 aliquot of each other treatment. Cells were washed twice with cell staining buffer (1% FBS in PBS), and then resuspended in 95 μL 1X Annexin V binding buffer (ThermoFisher) containing Vybrant® Cell Cycle Ruby dye (ThermoFisher) and Brilliant Violet-421™-Annexin V (BioLegend) as per manufacture’s instruction. For combination studies, cells were stained with 1X Annexin V binding buffer (Invitrogen) with 5 μM Sytox® Red dead cell stain (Life Technologies) and Annexin V Alexa Flour™488 conjugate (1:20 dilution) (ThermoFisher). Instrument controls included Sytox® only, Annexin V only, or unstained sample. Samples were then incubated in the dark at room temperature for 15 min and diluted 1:10 in PBS prior to analysis. Flow cytometric analysis was performed at the University of Arizona Translational Flow Cytometry Laboratory on a FACSCanto flow cytometry system (BD Biosciences) or Guava® easyCyte™ flow cytometer (Millipore). Acquired data was analyzed using FlowJo analysis software.

Montoya J.J., Turnidge M.A., Wai D.H., Patel A.R., Lee D.W., Gokhale V., Hurley L.H., Arceci R.J., Wetmore C, & Azorsa D.O. (2019). In vitro activity of a G-quadruplex-stabilizing small molecule that synergizes with Navitoclax to induce cytotoxicity in acute myeloid leukemia cells. BMC Cancer, 19, 1251.

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Sytox red dead cell stain

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Close spelling variants of this product

62 protocols using sytox red dead cell stain

Dissociation and Sorting of Tumor Cells

Fluorescence-Activated Cell Sorting of Zebrafish HSPC

Quantifying Cellular Uptake and Localization

Fluorescent Embryo Cell Sorting

ALDH-positive Cell Isolation and Sorting

Fluorescent Embryo Cell Sorting

Multiparametric Flow Cytometry of Tumor Microenvironment

Multiparametric Flow Cytometry of Tumor Microenvironment

ALDEFLUOR Assay for ALDH Detection

Apoptosis Quantification by Flow Cytometry

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