7 aminoactinomycin d

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

7-aminoactinomycin D is a fluorescent dye used in flow cytometry to detect and quantify apoptosis and cell cycle analysis. It binds to DNA and emits fluorescence upon binding, allowing for the identification of cells with compromised cell membranes.

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Lab products found in correlation

Facsaria 2, by BD (9 mentions) Facsdiva software, by BD (7 mentions) Facsaria, by BD (6 mentions) Facscanto 2, by BD (6 mentions) Click it edu alexa fluor 488 flow cytometry assay kit, by Thermo Fisher Scientific (6 mentions) Facsaria 3, by BD (5 mentions) Lsrfortessa, by BD (5 mentions) Facscanto 2 flow cytometer, by BD (5 mentions) Dnase 1, by Merck Group (4 mentions) Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (4 mentions) Kaluza software, by Beckman Coulter (4 mentions) Facscalibur, by BD (4 mentions) Lsrii flow cytometer, by BD (3 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions) 7 aad, by BD (3 mentions) Cytoflex flow cytometer, by Beckman Coulter (3 mentions) Moflo astrios eq, by Beckman Coulter (3 mentions) Gallios 2 5 3 flow cytometer, by Beckman Coulter (3 mentions) Bovine serum albumin (bsa), by Merck Group (3 mentions) Cytofix cytoperm kit, by BD (3 mentions)

Spelling variants of this product

7-aminoactinomycin D (7-AAD) (201 citations) Annexin V-APC/7-aminoactinomycin D (5 citations) 7-Aminoactinomycin D (7-AAD) Viability Staining Solution (3 citations) 7-Aminoactinomycin D (7-AAD) expression (2 citations) 7-aminoactinomycin D (7-ADD) (2 citations) 7-aminoactinomycin D (7AAD) live/dead dye (2 citations) 7-Aminoactinomycin D (7-AAD) viability dye (2 citations)

148 protocols using 7 aminoactinomycin d

Multiparameter Flow Cytometry Analysis

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The following antibodies were used: FOXP3-PECy5 (clone FJK-16s; eBioscience), H-2Kb-APC, CD4-e450, CD3-PECy7, B220-APCCy7 and CD45.2 PE (BD Pharmingen). Cell viability was determined using 7-Aminoactinomycin D (life technologies) or Fixable Viability Stain 510 (BD Horizon). Cells were analyzed on either a FACScan (BD Biosciences) or Gallios cytometer (Beckman coulter).

Cooper M.L., Choi J., Karpova D., Vij K., Ritchey J., Schroeder M.A, & DiPersio J.F. (2017). Azacitidine mitigates GvHD via differential effects on the proliferation of T effectors and nTregs in vivo. Journal of immunology (Baltimore, Md. : 1950), 198(9), 3746-3754.

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Cell Viability Assay Using Flow Cytometry

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Cell viability assays were performed in 96-well plates, with 6 × 10⁴ cells in 200 μl. Cells were harvested by centrifuging the 96-well plate in a plate spinner centrifuge at 500 g for 5 minutes. Cells were incubated in 1 μg/ml 7-aminoactinomycin D (Life Technologies) in Hank's Balanced Salt Solution (HBSS; Life Technologies) supplemented with 2.5% bovine serum albumin for 10 min at room temperature. Cell fluorescence was assessed using a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA). Analysis of the data was completed using FlowJo Software v10.0.7 (TreeStar, Ashland, OR).

Lee J.S., Tang S.S., Ortiz V., Vo T.T, & Fruman D.A. (2015). MCL-1-independent mechanisms of synergy between dual PI3K/mTOR and BCL-2 inhibition in diffuse large B cell lymphoma. Oncotarget, 6(34), 35202-35217.

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PBMC Immunophenotyping by Flow Cytometry

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Briefly, PBMCs were washed and pelleted. Each PMBC sample was labelled with an antibody mix (anti-CD16-FITC, anti-CD25-PE, anti-CD14-PE-Cy7, anti-CD19-APC, and anti-CD3-APC-Cy7, all from BD, and 7-aminoactinomycin D from Life Technologies) and incubated for 20 min. Next, cells were washed and acquired in a Guava EasyCyte 8HT flow cytometer (Millipore, Merk, Madrid, Spain). Single staining and isotype controls were used to set the gating strategy. The percentage of each cell population of PBMCs (T, B, NK cells, and monocytes), their activation, and cell viability were analyzed. All conditions were studied in duplicate and the experiment was repeated twice.

Sáenz-Cuesta M., Alberro A., Muñoz-Culla M., Osorio-Querejeta I., Fernandez-Mercado M., Lopetegui I., Tainta M., Prada Á., Castillo-Triviño T., Falcón-Pérez J.M., Olascoaga J, & Otaegui D. (2018). The First Dose of Fingolimod Affects Circulating Extracellular Vesicles in Multiple Sclerosis Patients. International Journal of Molecular Sciences, 19(8), 2448.

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Haematopoietic Induction of iPSCs

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Haematopoietic induction of iPSCs was assessed using CD34‐PE (345802; BD Biosciences) and CD45‐FITC (A07782; Beckman Coulter) antibodies. Dead cells were excluded using 7‐aminoactinomycin‐D (Life Technologies). Flow cytometry was performed using a LSRII flow cytometer (BD Biosciences). Cell sorting was done using a fluorescence‐activated cell sorting (FACS) Aria instrument (BD Biosciences). Analyses were performed using FlowJo (TreeStar).

Olofsen P.A., Bosch D.A., de Looper H.W., van Strien P.M., Hoogenboezem R.M., Roovers O., van der Velden V.H., Bindels E.M., De Pater E.M, & Touw I.P. (2022). Truncated CSF3 receptors induce pro‐inflammatory responses in severe congenital neutropenia. British Journal of Haematology, 200(1), 79-86.

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Isolation and Purification of Siglec-F+ Murine Intestinal Cells

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Freshly isolated small intestines of BC57BL/6 mice were flushed with PBS and incised along their length. The tissue was then incubated in 30mM EDTA (Sigma) in HBSS pH 7.4 (Life Technologies) on ice, and transferred in DMEM (Life Technologies) supplemented with 10% FBS (Sigma). Vigorous shaking yielded epithelial fraction that was then incubated with 100ul of Dispase (BD Biosciences) in 10 ml of HBSS, supplemented with 100ul of DNAse I at 2000 Kunitz (Sigma). Single cell preparation obtained by filtration on a 30 uM mesh was incubated with a Phycoerythrin Rat anti-mouse Siglec-F antibody for 30 minutes at 4°C (BD pharmigen 552126), washed with HBSS and resuspended in appropriate volume of HBSS pH 7.4 supplemented with 5% FBS before staining with 7-AminoActinomycin D (Life Technologies) to exclude dead cells. Siglec-F⁺ live cells were sorted using a FACSAria (Becton Dickinson).

Gerbe F., Sidot E., Smyth D.J., Ohmoto M., Matsumoto I., Dardalhon V., Cesses P., Garnier L., Bruschi M., Harcus Y., Taylor N., Maizels R.M, & Jay P. (2016). Intestinal epithelial tuft cells regulate type 2 mucosal responses required for expulsion of helminth parasites. Nature, 529(7585), 226-230.

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Transient Notch Overexpression in SCLC

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Mouse (KP1) and human (NJH29, NCI-H82 and NCI-H187) SCLC cell lines were grown in RPMI-1640 media supplemented with 10% bovine growth serum (BGS) (Fisher Scientific) and penicillin-streptomycin-glutamine (Gibco), as described before⁵⁰. KP1, NJH29 were generated at Stanford. NCI-H82 and NCI-H187 were purchased from ATCC. These cells grow as suspension spheres or aggregates in culture. All cell lines were maintained at 37 °C in a humidified chamber with 5% CO₂. All cell lines tested negative for mycoplasma infection. For transient expression of Notch ICD, cells were trypsinized and transfected with either MigR1-IRES-GFP (Ctrl) or MigR1-Notch1-ICD-IRES-GFP (NICD) using Lipofectamine 2000 (Life Technologies). The plasmids were gifts from W.S. Pear (University of Pennsylvania, Philadelphia). Then 48 h after transfection, cells were trypsinized and resuspended in phosphate-buffered saline (PBS) containing 10% BGS and 1 µg ml⁻¹ 7-aminoactinomycin D (Life Technologies) that labels dead cells. Live GFP⁺ cells were then sorted for subsequent experiments using a BD FACSAria fluorescence-activated cell sorting (FACS) machine.

George J., Lim J.S., Jang S.J., Cun Y., Ozretić L., Kong G., Leenders F., Lu X., Fernández-Cuesta L., Bosco G., Müller C., Dahmen I., Jahchan N.S., Park K.S., Yang D., Karnezis A.N., Vaka D., Torres A., Wang M.S., Korbel J.O., Menon R., Chun S.M., Kim D., Wilkerson M., Hayes N., Engelmann D., Pützer B., Bos M., Michels S., Vlasic I., Seidel D., Pinther B., Schaub P., Becker C., Altmüller J., Yokota J., Kohno T., Iwakawa R., Tsuta K., Noguchi M., Muley T., Hoffmann H., Schnabel P.A., Petersen I., Chen Y., Soltermann A., Tischler V., Choi C.M., Kim Y.H., Massion P.P., Zou Y., Jovanovic D., Kontic M., Wright G.M., Russell P.A., Solomon B., Koch I., Lindner M., Muscarella L.A., la Torre A., Field J.K., Jakopovic M., Knezevic J., Castaños-Vélez E., Roz L., Pastorino U., Brustugun O.T., Lund-Iversen M., Thunnissen E., Köhler J., Schuler M., Botling J., Sandelin M., Sanchez-Cespedes M., Salvesen H.B., Achter V., Lang U., Bogus M., Schneider P.M., Zander T., Ansén S., Hallek M., Wolf J., Vingron M., Yatabe Y., Travis W.D., Nürnberg P., Reinhardt C., Perner S., Heukamp L., Büttner R., Haas S.A., Brambilla E., Peifer M., Sage J, & Thomas R.K. (2015). Comprehensive genomic profiles of small cell lung cancer. Nature, 524(7563), 47-53.

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Phagocytic Activity of SLN LCs

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Phagocytic activity of SLN LCs was examined with FITC-Dextran assay [21 (link)]. SLN cells were collected as described above and re-suspended in complete medium. Briefly, each SLN sample was divided into two aliquots: the first, indicated as t = 0, was processed immediately; the latter, indicated as t = 24, was cultured for 24 h with the following pro-inflammatory cytokines: IL-1β (10 ng/ml), IL-6 (1000 U/ml) and TNF-α (10 ng/ml), all from R & D Systems (Minneapolis, MN), for phagocytic activity inhibition induced by maturation. Both samples were further incubated at 0 °C (on ice) and 37 °C for 10 min and then pulsed with 40,000 m.w. FITC-conjugated Dextran (1 mg/ml; Life Technologies) for 45 min, both at 0 °C and 37 °C. After washing steps with cold PBS, cells were labelled with anti Langerin PE-conjugated mAbs for 15 min at room temperature without fixation and permeabilization. To exclude dead cells, 7-aminoactinomycin D (7-AAD; Life Technologies) was used. Cells were then analyzed by flow cytometry, using FACS Canto and FACSDiva software, version 6.0 (Becton Dickinson Immunocytometry Systems, San Jose, CA). Results are expressed as mean fluorescence intensity (MFI). Cells incubated at 0 °C represent assay internal control, with no phagocytic activity.

Romoli M.R., Gennaro P.D., Gerlini G., Sestini S., Brandani P., Ferrone S, & Borgognoni L. (2017). High Antigen Processing Machinery component expression in Langerhans cells from melanoma patients’ sentinel lymph nodes. Cellular immunology, 320, 29-37.

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Irisin-Induced Apoptosis in PC-3 Cells

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Apoptotic potential for irisin against PC-3 was evaluated using flow cytometry. PC-3 cells were treated with ≈2 × IC50 (100 nmol/L) irisin for 24, 48, and 72 h. Cells were then harvested, suspended in 500 μL Annexin binding buffer (ABB), and either incubated with 5 μL/mL Annexin-APC (Annexin-V conjugated to allophycocyanin, Life Technologies, Grand Island, NY, USA) for 15 min or stained by adding 1 μL/mL of 7-aminoactinomycin D (7-AAD) Life Technologies (Grand Island, NY, USA) and incubated in ice for 45 min. Treated samples and controls were analyzed using FACSCanto-II (BD-BioSciences, San Jose, CA, USA) at the College of Pharmacy, King Saud University. APC was excited with a 635 nm laser and detected at 660 nm for Annexin-V and with a 488 nm laser and detected at 660 nm for 7-AAD Assay.

Alshanqiti K.H., Alomar S.F., Alzoman N, & Almomen A. (2023). Irisin Induces Apoptosis in Metastatic Prostate Cancer Cells and Inhibits Tumor Growth In Vivo. Cancers, 15(15), 4000.

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Isolation of Murine Hematopoietic Stem Cells

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Mice were sacrificed by dislocation of the neck. BM cells were isolated from the tibia, femur and sternum of both hind legs, by crushing bones in PBS (Sigma) supplemented with 2% Fetal Calf Serum (FCS (Sigma) or STEMCELL Technologies (SCT)). Samples were filtered through 20‐μm sterile filters before further processing. Red cell lysis was performed using ammonium chloride (NH₄Cl, SCT) and HSPC were enriched by magnet separation using EasySep Mouse Hematopoietic Progenitor Cell Enrichment Kit (SCT). ESLAM Sca‐1⁺ cells were isolated by fluorescence‐activated cell sorting (FACS) using CD45 BV421 (Clone 30‐F11, Biolegend), CD150 PE/Cy7 (Clone TC15‐12F12.2, Biolegend), CD48 APC (Clone HM48‐1, Biolegend), Sca‐1 BV605 (Clone D7, Biolegend), EPCR PE (Clone RMEPCR1560, SCT), and 7‐Aminoactinomycin D (7AAD; Life Technologies). The cells were sorted on an Influx (BD) using the following filter sets 530/40 (for Fgd5), 585/29 (for PE), 670/30 (for APC), 460/50 (for BV421), 670/30 (for 7AAD), and 610/20 (for BV605). When single HSCs were required, the single‐cell deposition unit of the sorter was used to place 1 cell per well into 96‐well plates, each well having been preloaded with 50 μl or 100 μl medium. E14.5 FLs were prepared and stained as above and analyzed as above.

Che J.L., Bode D., Kucinski I., Cull A.H., Bain F., Becker H.J., Jassinskaja M., Barile M., Boyd G., Belmonte M., Zeng A.G., Igarashi K.J., Rubio‐Lara J., Shepherd M.S., Clay A., Dick J.E., Wilkinson A.C., Nakauchi H., Yamazaki S., Göttgens B, & Kent D.G. (2022). Identification and characterization of in vitro expanded hematopoietic stem cells. EMBO Reports, 23(10), e55502.

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Immune Profiling of Tumor Microenvironment

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Fine-needle aspirates (FNAs) were taken with a 25G needle from anesthetized P815 tumor-bearing mice on days 1, 3, 5, 8, 11, 14, and 16 post-treatment, with individual mice receiving between two and five FNAs.23 (link) Briefly, aspirates were digested at 37°C for 20 min using 5 mg/mL Collagenase type I (Worthington Biochemical, UK), 1.5 mg/mL Hyaluronidase (Sigma-Aldrich), and 0.01 mg/mL DNAse I (Worthington Biochemical) in DMEM supplemented with 1% FCS. Samples were then stained and analyzed by flow cytometry (LSRII, Becton Dickinson, USA; FlowJo, Treestar, USA). Two stains were used to identify immune cell types. Both stains contained a P815 tumor marker anti-CD63 (NVG-2) PE, pan-immune cell marker anti-CD45.2 (104) Alexa Fluor 488, and 7-aminoactinomycin-D (Life Technologies) for live/dead cell discrimination, as well as: Stain A—anti-CD8α (53.6.7) APC Cy7 (BD Biosciences), anti-CD4 (GK1.5) PE-Cy7, anti-CD25 (PC61) Brilliant Violet 421, anti-CD19 (6D5) Brilliant Violet 510, and anti-Pan-NK (CD49b) (Dx5) APC. Stain B—anti-CD11b (M1/70) Brilliant Violet 510, anti-CD11c (N418) PE Cy7, anti-F4/80 (BM8) Brilliant Violet 421, anti-Ly6G/Ly6C (GR1) (RB6-8C5) APC Cy7, and anti-Siglec-F (E50-2440) Alexa Fluor 647 (BD Biosciences, USA). All antibodies were from Biolegend (California, USA) unless otherwise specified.

Carroll C.S., Andrew E.R., Malik L., Elliott K.F., Brennan M., Meyer J., Hintze A., Almonte A.A., Lappin C., MacPherson P., Schulte K.M., Dahlstrom J.E., Tamhane R., Neeman T., Herbert E.W., Orange M., Yip D., Allavena R, & Fahrer A.M. (2021). Simple and effective bacterial-based intratumoral cancer immunotherapy. Journal for Immunotherapy of Cancer, 9(9), e002688.

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