7 aminoactinomycin d 7 aad

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7-aminoactinomycin D (7-AAD) is a fluorescent dye used in flow cytometry applications. It is a DNA-binding agent that exhibits an increase in fluorescence intensity upon binding to DNA. 7-AAD is commonly used as a viability stain to detect dead or dying cells in a sample.

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Lab products found in correlation

Facscanto 2, by BD (9 mentions) Novocytetm 2000 flowcytometry system, by Agilent Technologies (9 mentions) Annexin 5, by BioLegend (8 mentions) Annexin 5 fitc, by BioLegend (8 mentions) Facsaria 2, by BD (7 mentions) Lsrfortessa, by BD (7 mentions) Mitotracker green, by Thermo Fisher Scientific (6 mentions) Facscalibur, by BD (5 mentions) Software, by FlowJo (5 mentions) Facscalibur flow cytometer, by BD (4 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (4 mentions) Cyan adp instrument, by Agilent Technologies (3 mentions) Facsaria, by BD (3 mentions) Annexin 5 fitc, by BD (3 mentions) Facscanto 2 flow cytometer, by BD (3 mentions) Annexin 5 apc, by BioLegend (3 mentions) Attune nxt flow cytometer, by Thermo Fisher Scientific (3 mentions) Ghost dye violet 510, by Cytek Biosciences (2 mentions) Accuri c6 cytometer, by BD (2 mentions) Annexin 5 binding buffer, by BioLegend (2 mentions)

129 protocols using 7 aminoactinomycin d 7 aad

Estradiol and 17β-Aminoestrogen Synthesis

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Estradiol (E2, 1,3,5(10)-estratrien-3,17β diol) and sulforhodamine B (SRB) were purchased from Sigma Aldrich (St. Louis, MO, USA). The 17β-AEs, prolame [17β-(3-hydroxy-1-propylamine)-1,3,5(10)-estratrien-3-ol)], butolame [17β-(3-hydroxy-1-butylamine)-1,3,5(10)-estratrien-3-ol)], and pentolame [17β-(5-hydroxy-1-pentolamine)-1,3,5(10)-estratrien-3-ol)], were prepared from estrone according to previously reported methods [21 (link),44 (link)]. G36 (4-(6-bromo-benzo[1,3]dioxol-5-yl)-8-isopropyl-3a,4,5,9b-tetrahydro-3H-cyclopenta quinoline) was synthesized and characterized according to the procedure reported by Denis et al. [43 (link)].
The compounds were synthesized following exactly the reported procedures [21 (link),44 (link)]. E2, the 17β-aminoestrogens, and G36 were dissolved in absolute ethanol and used as a stock solution (Merck, Darmstadt, Germany). All cell culture reagents and media, as well as the molecular biology reagents, were purchased from Gibco (Invitrogen Corporation, Waltham, MA, USA).
Additionally, 7-Aminoactinomycin D (7-AAD) was acquired from BioLegend, San Diego, CA, USA, Phosflow Fix Buffer I and Phosflow Perm Buffer III were purchased from Becton Dickinson (BD) Biosciences, San Jose, CA, USA), and the rabbit anti-phospho c-fos (SER32) monoclonal antibody was acquired from Cell Signaling Technology, Danvers, MA, USA.

Segovia-Mendoza M., Mirzaei E., Prado-Garcia H., Miranda L.D., Figueroa A, & Lemini C. (2022). The Interplay of GPER1 with 17β-Aminoestrogens in the Regulation of the Proliferation of Cervical and Breast Cancer Cells: A Pharmacological Approach. International Journal of Environmental Research and Public Health, 19(19), 12361.

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Wnt Signaling and T Cell Apoptosis

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The effect of TWS119-mediated Wnt signaling activation on apoptosis of different subsets of T cells was evaluated by flow cytometry. Briefly, activated CD8⁺ T cells were treated with 5 µM TWS119. Tumor necrosis factor-alpha (TNF-ɑ, 1 µg/mL; PeproTech, AF-200-04) was added at day 5. Cells were collected on day 6 and were first stained with the antibodies for surface markers to identify Tscm (CD45RA⁺CD62L⁺CD95⁺), T_CM (CD45RA^–CD62L⁺CD95⁺), T_EM (CD45RA^–CD62L^–CD95⁺), and effector memory RA (TEMRA) (CD45RA⁺CD62L^–CD95⁺). The cells were then washed twice with staining buffer and stained with anti-Annexin V antibody to detecting early apoptosis and 7-aminoactinomycin D (7-AAD; Biolegend, 640922) to detect late apoptosis.

Yan C., Chang J., Song X., Yan F., Yu W., An Y., Wei F., Yang L, & Ren X. (2019). Memory stem T cells generated by Wnt signaling from blood of human renal clear cell carcinoma patients. Cancer Biology & Medicine, 16(1), 109-124.

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T-cell Transfer-Induced Colitis in Mice

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Colitis was induced in C.B-17-scid/scid mice by adoptive transfer of CD45RB^high T cells. Mojosort Mouse CD 4 T Cell Isolation Kit (BioLegend, San Diego, CA, United States) and biotin-conjugated anti-mouse TER-119 (TER-119; BioLegend) were used to negatively select CD4⁺ T cells of splenocytes from C.B-17^+/+ mice. Negatively selected CD4⁺ T cells were labeled with Alexa Fluor 647-conjugated anti-mouse CD4 (RM-4-5; BioLegend), BV510-conjugated anti-mouse CD45 (30-F11; BioLegend), and BV421-conjugated anti-mouse CD45RB (16A; BD Horizon, San Jose, CA, United States). Dead cells were labeled with 7-aminoactinomycin D (7-AAD) (BioLegend). CD45⁺CD4⁺CD45RB^high live T cells were isolated by FACS Aria III (BD Biosciences San Jose, CA, United States). The C.B-17-scid/scid recipients were each injected with 2 × 10⁵ cells via the tail vein and transferred to conventional conditions in the animal facilities at Keio University. Mice were fed with 0.015% NEt-3IB-containing MF diet or control MF diet for 7 weeks. The body weights of the mice were measured every week, and the fecal diarrhea score was also measured from week 4. The diarrhea score were assessed as follows: normal stool (0), slightly soft (1), soft but formed (2), not formed (3), and liquid stool (4) (Kjellev et al., 2006 (link)). The mice were humanely killed at week 7.

Matsumoto R., Takahashi D., Watanabe M., Nakatani S., Takamura Y., Kurosaki Y., Kakuta H, & Hase K. (2021). A Retinoid X Receptor Agonist Directed to the Large Intestine Ameliorates T-Cell-Mediated Colitis in Mice. Frontiers in Pharmacology, 12, 715752.

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Quantification of Tfh and GC B Cells in Immunized Mice

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Mice were immunized subcutaneously (s.c.) with a total protein dose corresponding to 5 μg of DS-Cav1 antigen on day 0. All proteins were formulated in PBS in a 1:1 ratio with AddaVax adjuvant (InvivoGen). Six to seven mice per group were sacrificed at day 7 after priming, and draining lymph nodes were collected and converted to cell suspensions. The total number of cells in each suspension was counted by trypan blue exclusion with a hemocytometer. One-fifth of the cell suspension was used for FACS counting. The total number of Tfh and GC B cells (plotted in Figure 6) was calculated by multiplying the percentage of detected Tfh or GC B cells by the total number of cells in each suspension. The following antibodies were used to identify Tfh cells and GC B cells: CD4 (RM4-5), PD-1 (RMP1-30), B220 (RA3-6B2) (Thermo Fisher Scientific), CD3ε (17A2), CD45.1 (A20), ICOS (7E.17G9) (BioLegend), CXCR5 (2G8), CD19 (1D3), FAS (Jo2) (BD Biosciences), and Peanut Agglutinin (PNA) (Vector Laboratories). Dead cells were excluded from counting by staining with 7-aminoactinomycin D (7-AAD; BioLegend). Samples were acquired on a BD LSR Fortessa instrument and analyzed using the FlowJo software program (TreeStar).

Marcandalli J., Fiala B., Ols S., Perotti M., de van der Schueren W., Snijder J., Hodge E., Benhaim M., Ravichandran R., Carter L., Sheffler W., Brunner L., Lawrenz M., Dubois P., Lanzavecchia A., Sallusto F., Lee K.K., Veesler D., Correnti C.E., Stewart L.J., Baker D., Loré K., Perez L, & King N.P. (2019). Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle Vaccine for Respiratory Syncytial Virus. Cell, 176(6), 1420-1431.e17.

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Murine Splenocyte Immune Profiling

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Mouse splenocytes were stained with Fc-receptor blocking antibodies (clone 2.4G2, 1:100 dilution, BD Biosciences) for 10 min on ice to block non-specific staining. For surface staining, cells were washed once with PBS containing 2% heat-inactivated fetal bovine serum (FBS, Gibco) and stained in an appropriately diluted antibody solution for 40 min at 4 C. Dead cells were excluded after staining with 7-amino-actinomycin D (7-AAD, BioLegend). For intracellular staining of cytokines, PMA and ionomycin stimulated cells were washed once after surface staining and permeabilized using Cytofix/Cytoperm (BD Biosciences) for 40 min on ice. The antibodies were diluted in Perm/Wash Buffer (BD), and stained in an appropriately diluted antibody solution for 1 h at 4 C. For intranuclear staining of transcription factors, cells were washed once after surface staining and permeabilized using Foxp3/Transcription Factor Staining Buffer Set (eBioscience) for 40 min on ice. The specific antibodies were diluted in Fixation/Permeabilization buffer (eBioscience) and incubated for 1 h at 4 C. Data were collected on a BD FACS Aria III flow cytometer (BD Biosciences) and analyzed using FlowJo (BD Biosciences) software. Antibody information is presented in Table 2.

Zheng T., Fan M., Wei Y., Feng J., Zhou P., Sun X., Xue A., Qin C.X, & Yu D. (2021). Huangbai Liniment Ameliorates Skin Inflammation in Atopic Dermatitis. Frontiers in Pharmacology, 12, 726035.

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Measuring CAR Expression in Cells

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Example 5

To measure CAR expression, 0.5 million cells were suspended in 100 μl of buffer (PBS containing 0.5% BSA) and incubated on ice with 1 μl of human serum (Jackson Immunoresearch, West Grove, Pa.) for 10 min. Then 1 μl of allophycocyanin (APC)-labeled anti-CD3 (eBioscience, San Diego, Calif.), 2 μl of 7-aminoactinomycin D (7-AAD, BioLegend, San Diego, Calif.), and 2 μl of biotin-labeled polyclonal goat anti-human-F(ab)₂antibodies (Life Technologies) that detect EGFR scFv, Meso scFv, or CD19 scFv, or biotin-labeled normal polyclonal goat IgG antibodies (Life Technologies) were to detect CAR expression. The cells were rinsed with 3 ml of buffer, then suspended in buffer and acquired on a FACSCalibur (BD Biosciences). Cells were analyzed first for light scatter versus 7-AAD staining, then the 7-AAD⁻live gated cells were plotted for CD3 staining versus F(as)₂staining or isotype control staining.

US11332513B2. Chimeric antigen receptors having GITR intracellular domain as co-stimulatory domain (2022-05-17). ProMab Biotechnologies, Inc. [US], Forevertek Biotechnology Co., Ltd [CN], The General Hospital Corporation [US]. Inventors: Lijun Wu [US], Marcela V. Maus [US], Vita Golubovskaya [US].

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Cell Surface Marker Identification and Sorting

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Pacific Blue-conjugated anti-mouse CD24 or PerCP/Cy5.5-conjugated anti-human CD24 (Biolegend, San Diego, CA, USA) were used for cell surface staining of the cells. 7-amino actinomycin D (7-AAD, Biolegend) was used to gate live cells. Cells were stained at a concentration of 5 × 10⁶ cells/ml of fluorescence-activated cell sorting (FACS) buffer (PBS containing 0.1 % bovine serum albumin [BSA]) for 20 minutes on ice in the dark, after which, the cells were washed twice and resuspended in FACS buffer containing 7-AAD. Stained cells were analyzed using the CyAn ADP Instrument (Dako-Cytomation, Glostrup, Denmark) and the FlowJo 7.25 analysis software (Tree Star, Ashland, OR, USA). Flow cytometry-based cell sorting for CD24^- IGF1R-KD and CD24⁺ IGF1R-KD cells was performed using FACSAria (BD Biosciences, San Jose, CA, USA).

Rostoker R., Ben-Shmuel S., Rashed R., Shen Orr Z, & LeRoith D. (2016). CD24 cell surface expression in Mvt1 mammary cancer cells serves as a biomarker for sensitivity to anti-IGF1R therapy. Breast Cancer Research : BCR, 18, 51.

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Flow Cytometric Analysis of DR4/DR5 Expression and Cell Death

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For both assays, a FACSCalibur flow cytometer was used to detect the cells and a computer with the CellQuestPro software (BD Biosciences, Franklin Lakes, NJ, USA) was used to collect the data. Afterwards, Flow Jo software (Tree Star Inc., San Francisco, CA, USA) was used to analyze them.
DR4 and DR5 surface expression was determined as follows: 1 × 10⁵ tumor cells were incubated with specific antibodies for DR4, DR5, DcR1, or DcR2. An additional isotype control antibody was also used (all of them conjugated with PE and purchased from eBioscience, San Diego, CA, USA). Staining conditions were: tumor resuspension in PBS containing 5% FBS with the corresponding antibody for 30 min at 4 °C. Then, cells were washed once, resuspended in 300 µL with PBS and analyzed in the FACSCalibur.
For the measurement of cell death induction, 1 × 10⁵ tumor cells were incubated for 15 min with both Annexin-V-FITC (Immunostep, Salamanca, Spain), which recognizes exposed phosphatidylserine, and 7-aminoactinomycin D (7-AAD, Biolegend, San Diego, CA, USA), which binds DNA. The mixture was resuspended in annexin-binding buffer (140 mM NaCl, 2.5 mM CaCl₂, 10 mM HEPES/NaOH, pH 7.4). After staining, cells were washed once with PBS and their fluorescence was measured by flow cytometry.

Marco-Brualla J., de Miguel D., Martínez-Lostao L, & Anel A. (2023). DR5 Up-Regulation Induced by Dichloroacetate Sensitizes Tumor Cells to Lipid Nanoparticles Decorated with TRAIL. Journal of Clinical Medicine, 12(2), 608.

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Apoptosis Assay of Cell Cultures

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One million cells from each culture were kept in the dark for 30 min at 4° with Ghost Dye Violet 510 (Tonbo Biosciences, San Diego, CA). The cells were washed in FACS buffer, fixed in PBS containing 2% paraformaldehyde, and analyzed on a BD FACS-CantoII (Becton Dickinson, San Jose, CA). The data collected were analysed using Flow Jo software (FlowJo, Ashland, OR).The gating strategy used was lymphocytes (FSC-A × SSC-A) and then singlets (FSC-A × FSC-H) before focusing on viability. For measurement of apoptosis, 1 million cells from each time point were incubated in the dark in annexin binding buffer with 5 μl each of annexin V–FITC and 7–aminoactinomycin D (7-AAD) (both from BioLegend) for 10 min at room temperature. A total of 0.5 ml of annexin binding buffer was then added to each culture and immediately analysed on an Accuri C6 cytometer (BD Biosciences). The gating strategy was as described above.

, & Takada S. (1999). Gō-ing for the prediction of protein folding mechanisms. Proceedings of the National Academy of Sciences of the United States of America, 96(21), 11698-11700.

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PBMC Isolation and Phenotypic Analysis

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Eight to sixteen milliliters of whole blood was drawn and PBMCs were isolated with BD Vacutainer CPT (Becton Dickinson, Franklin Lake, NJ, USA), containing 1.0 ml of 0.1 M sodium citrate, 3 g of polyester gel, and 2.0 ml of polysaccharide sodium diatrizoate. After centrifugation at 1500 × g for 25 minutes, PBMCs on the gel phase were harvested, and washed twice with RPMI-1640 medium (Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% bovine albumin. Isolated PBMCs were incubated with specific fluorescence-labeled monoclonal antibodies (mAbs) at 4°C for 30 min. Anti-human mAbs including CD3 (FITC, BD), CD56 (PE-Cy7, BD), CD4 (APC, BD), CD8 (APC-Cy7, BD), 7-amino-actinomycin D (7-AAD) and NKG2D (PE, clone 1D11, BioLegend, San Diego, CA, USA) were used. Dead cells were excluded from assessment by 7-AAD. Irrelevant anti-rat isotype antibodies (BD) were used to assess background fluorescence. Stained cells were analyzed using FACS Canto II (BD), and the data was analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA). Gating strategies are illustrated in supporting S1 Fig NKG2D expression is defined as the percentage of NKG2D-positive cells compared to isotype controls, as illustrated in Fig 1B.

Chu P.S., Ebinuma H., Nakamoto N., Sugiyama K., Usui S., Wakayama Y., Taniki N., Yamaguchi A., Shiba S., Yamagishi Y., Wakita T., Hibi T., Saito H, & Kanai T. (2015). Genotype-Associated Differential NKG2D Expression on CD56+CD3+ Lymphocytes Predicts Response to Pegylated-Interferon/ Ribavirin Therapy in Chronic Hepatitis C. PLoS ONE, 10(5), e0125664.

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