7 aad solution

Manufactured by BD

Sourced in United States

7-AAD solution is a fluorescent dye that binds to DNA and is used in flow cytometry applications to detect and analyze cells. It is a common reagent used for cell viability and cell cycle analysis.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Anti cd3 apc efluor780, by Thermo Fisher Scientific (1 mentions) Anti cd8 vioblue, by Miltenyi Biotec (1 mentions) 96 well plate, by Greiner (1 mentions) Macsquant flow cytometer, by Miltenyi Biotec (1 mentions) Accuri c6 device, by BD (1 mentions) T9284, by Merck Group (1 mentions) Ercc rna spike in mix, by Thermo Fisher Scientific (1 mentions) Triton x 100, by Merck Group (1 mentions) Facsaria 3, by BD (1 mentions) Facsaria 2, by BD (1 mentions) Facsaria 3 cell sorter flow cytometer, by BD (1 mentions) Apc conjugated annexin 5, by BD (1 mentions) Annexin 5 binding buffer, by BD (1 mentions) Annexin 5 apc, by BD (1 mentions) Cellquest software, by BD (1 mentions)

Spelling variants of this product

7-AAD (1194 citations) 7-AAD staining solution (13 citations) 7-AAD viability staining solution (13 citations) 7-Amino-actinomycin D (7-AAD) staining solution (5 citations)

9 protocols using 7 aad solution

Fas-Induced Cell Death Kinetics

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Jurkat cells were seeded in 18 wells of 6-well plates (5 × 10⁵ cells/well) in RPMI medium with 5% FBS. The next day, the cells were washed once with phosphate buffered saline (PBS) and re-suspended in 1 mL of RPMI medium containing 12.5 ng/mL of anti-human Fas IgM (clone CH11). At each time point (0, 0.5, 1, 2, 4, and 8 h, N = 3 for each time point), we harvested 500 μL of cell suspension, centrifuged it at 100 g for 5 min, transferred the medium to fresh microfuge tubes, and froze both the media and cell pellets separately at − 80 °C until the assays for LDH, n-DNA, fortilin, Cyt C, and fCK-18, were performed. For 7-AAD staining, we added 20 μL of 7-AAD solution (BD Pharmingen) to 400 μL of cell suspension and incubated it for 10 min at room temperature, shielded from light. We counted total and 7-AAD-positive cells under the fluorescence microscope as described previously [26] (link). The integrity of the plasma membrane of the cells with positive 7-AAD signal is compromised. At least 200 cells were counted and the 7-ADD index was calculated as (the number of 7-AAD-positive cells) / (the number of total cells) ∗ 100.

Sinthujaroen P., Wanachottrakul N., Pinkaew D., Petersen J.R., Phongdara A., Sheffield-Moore M, & Fujise K. (2014). Elevation of serum fortilin levels is specific for apoptosis and signifies cell death in vivo. BBA Clinical, 2, 103-111.

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Quantifying CD69 Expression in Jurkat T Cells

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J.RT3-T3.5 2B9 cells (1 million) were transduced by direct addition of 1 mL HEK293T supernatant (see S1 Methods) and incubated for 72 h. Transduced J.RT3-T3.5 2B9 cells and co-cultures of these cells with antigen-presenting cells (100,000 cells each) were incubated in 96-well plates (Greiner Bio-one) for approximately 16 h. Supernatants containing Jurkat cells were removed and the cells pelleted. Cells were washed once with PBS with 0.1% fetal bovine serum (Gibco) before incubation with anti-CD69-PE (12-0699-42, Thermo Fisher Scientific), anti-CD3-APC-eFluor780 (47-0038-42, Thermo Fisher Scientific), and anti-CD8-VioBlue (130-110-684, Miltenyi Biotec) for 30 min at 4°C. Cells were pelleted and washed once with PBS with 0.1% fetal bovine serum. Cells were pelleted and resuspended in 7-AAD solution (51–6898, BD Pharmingen) before data were acquired on a Miltenyi MACSQuant flow cytometer. Data were analyzed using FlowJo v10.6.0 (FlowJo LLC). CD69-positive events were quantified upon gating on lymphocytes (SSC-A vs FSC-A), singlets (FSC-A vs FSC-H), live cells (7-AAD vs FSC-A), and CD8- and CD3-positive cells (S1 Fig).

Hiscox M.J., Wasmuth A., Williams C.L., Foot J.N., Wiedermann G.E., Fadda V., Boiani S., Cornforth T.V., Wikiert K.A., Bruton S., Cartwright N., Anderson V.E., Barnes C.S., Vieira J.V., Birch-Machin I., Gerry A.B., Miller K, & Pumphrey N.J. (2024). Selection, engineering, and in vivo testing of a human leukocyte antigen–independent T-cell receptor recognizing human mesothelin. PLOS ONE, 19(4), e0301175.

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Propolis Extracts and Cell Cycle

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To determine if propolis extracts can induce changes in DNA content in different phases of the cell cycle, increasing the proliferation of HGnF cells, the 7-amino actinomycin D (7-AAD) [78 (link)] method was used. Thus, 2 × 10⁴ cells/well were seeded in 24-well plates. After 24 h of culture stabilization, the cells were treated with propolis extracts, demonstrating lower toxicity levels at concentrations of 1.0, 2.5, and 5.0 µg/mL for 48 h. The culture medium was removed, and the cells were dissociated with trypsin–EDTA solution (0.25–0.5 mM) for 5 min at 37 °C and washed twice with PBS. The cell pellet was fixed with 70% ethanol and stained with 25 mg/mL 7-AAD solution (BD Pharmingen, San Diego, CA, USA) for 15 min at 20–25 °C in the dark. Cell cycle analysis was performed by flow cytometry on the BD Accuri C6 device using a 488–647 nm laser. The results were analyzed using Modfit LT software version 5.0 and graphed using GraphPad Prism 7.0 software. Unstimulated cells were used as negative controls. For this study, three independent experiments were performed in duplicate.

Buitrago D.M., Perdomo S.J., Silva F.A., Cely-Veloza W, & Lafaurie G.I. (2024). Physicochemical Characterization, Antioxidant, and Proliferative Activity of Colombian Propolis Extracts: A Comparative Study. Molecules, 29(7), 1643.

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Single-cell RNA sequencing of mouse islet cells

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After treatment of the dissociated islets with the mitogens (Mouse islet isolation and cell culture), islet cells were incubated with Accutase for 15 min at 37 °C. Detached islet cells were resuspended in PBS containing 2% FBS and 5 mM EDTA. Dead cells were stained with a 7-AAD solution (559925, BD Biosciences) 5 min before flow cytometry cell sorting analysis and cell sorting using a BD FACSAria III (BD Biosciences) with software FACSDiva version 8.0.1. Flow cytometry was performed using a ceramic nozzle with a size of 100 μm. Cell doublets were excluded by gating singlets on FSC-W and SSC-W. By using a single-cell discrimination mask, viable individual islet cells were sorted and collected in FrameStar 384-well plates (4TI-0384/C, Saveen Werner) containing 2.3 μl lysis buffer, which included 0.4% Triton X-100 (T9284, Sigma-Aldrich), one part RNase inhibitor (2313A, Clontech), 2.5 μM Smart dTVN30 oligonucleotides^{37 (link)}, 4 mM dNTP (R1122, Thermo Fisher Scientific) and the ERCC RNA Spike-In mix, diluted 1:40,000 (4456740, Thermo Fisher Scientific). Sample plates were kept at −80 °C for future preparation of Smart-seq2 cDNA libraries.

Charbord J., Ren L., Sharma R.B., Johansson A., Ågren R., Chu L., Tworus D., Schulz N., Charbord P., Stewart A.F., Wang P., Alonso L.C, & Andersson O. (2021). In vivo screen identifies a SIK inhibitor that induces β cell proliferation through a transient UPR. Nature metabolism, 3(5), 682-700.

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Cell Harvesting and FACS Sorting

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Cells were harvested by trypsinization for 5 min at 37 °C and stained with 7-AAD solution (BD Pharmingen, Cat# 559925, 1:200 with DMEM) for 10 min at room temperature to exclude dead cells. Cells were fixed with CellCover (AL Anacyte Laboratories, Cat# 800-125) and sorted using FACS Aria 2 (BD Biosciences) (Figure S2A).

Narita S., Unno K., Kato K., Okuno Y., Sato Y., Tsumura Y., Fujikawa Y., Shimizu Y., Hayashida R., Kondo K., Shibata R, & Murohara T. (2022). Direct reprogramming of adult adipose-derived regenerative cells toward cardiomyocytes using six transcriptional factors. iScience, 25(7), 104651.

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Annexin V Apoptosis Assay Protocol

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Briefly, 3×10⁶ cells were plated, cultured for overnight and then treated with 20 µM of ABT-737 for 24 h. Apoptosis was detected using APC-conjugated Annexin V and 7-Amin-Actinomycin D (7-AAD) (BD Biosciences, USA). At the end of ABT-737 treatment, cells were collected and washed twice in cold PBS, re-suspended in Annexin V‑binding buffer (#51-66121E, BD Biosciences, USA) at a concentration of 1×10⁶ cells/ml. One hundred μl of the cell suspension was incubated with 5 μl of APC Annexin V (#550475, BD Biosciences, USA) and 5 μl of 7-AAD solution (#51-68981E, BD Biosciences, USA) in dark at room temperature for 15 minutes. Following the addition of 400 μl binding buffer to each cellular sample at the end of incubation, cells were analyzed using FACSAria™ III Cell Sorter flow cytometer (BD Biosciences, USA). The flow cytometry results were analyzed using BD FlowJo software (BD Biosciences, USA).

Yang P., Xie J., Li Y., Lin H.P., Fenske W., Clementino M., Jiang Y., Yang C, & Wang Z. (2020). Deubiquitinase USP7-mediated MCL-1 up-regulation enhances Arsenic and Benzo(a)pyrene co-exposure-induced Cancer Stem Cell-like property and Tumorigenesis. Theranostics, 10(20), 9050-9065.

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CD8+ T-cell Cytotoxicity Assay Protocol

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Targets were CD8-depleted from isolated PBMCs of HLA class I matched HIV-seronegative donors as above. The targets were activated with PHA (5μg/mL) in the presence of IL-2 (100 U/mL) for 2 days. Activated matched or mismatched CD4 targets (5×10⁵ cells) were co-cultured with appropriate HLA CD8 T-cells (Effectors) from HIV-seronegatives for 24 hrs at four effector to target (E/T) ratios. Cells were surface-stained with anti-CD3-Pacific Blue and anti-CD4-Alexa780 (eBioscience) followed by staining with 0.25μg 7-AAD solution (BD Biosciences) for 20 mins to detect dead cells. Dead targets were measured by gating on 7-AAD⁺CD4⁺ T-cell populations, and samples with no effectors were used to set the background gates. Target cell death was determined by comparing the net percentage of 7-AAD+CD4+ T-cells in the presence of effectors relative to no effectors. Percent killing is calculated as follows: (% 7-AAD targets with effectors - %7-AAD targets without effectors/100- %7-AAD targets without effectors) × 100^{[19 (link)]}. All flow cytometry data was analyzed using FlowJo Version 9.4 software (TreeStar, San Carlos, CA).

SABBAJ S., SCANLON N., DU V.Y., WANG Y., TANG J., HUNTER E, & GOEPFERT P.A. (2016). Enhanced allogeneic cellular responses to mismatched HLA-B antigens results in more efficient killing of HIV infected cells. Journal of acquired immune deficiency syndromes (1999), 71(5), 493-497.

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Annexin V-PE/7-AAD Apoptosis Assay

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For the apoptosis assay, dissociated cells were washed with phosphate buffered saline (PBS, HyClone, South Logan, UT, USA) and resuspended in annexin-binding buffer at 1 × 10⁵ cells/100 μL. Thereafter, cell suspension was incubated for 15 min with 5 μL of Phycoerythrin (PE)-Annexin V and 5 μL of 7-AAD solution (BD Pharmingen, San Diego, CA, USA) at room temperature. After incubation, cells were analyzed by flow cytometry. The Data were analyzed with Cell Quest Software (BD bioscience, San Jose, CA, USA).

Lee J.H., Park S.Y., Hwang W., Sung J.Y., Cho M.L., Shim J., Kim Y.N, & Yoon K. (2020). Isoharringtonine Induces Apoptosis of Non-Small Cell Lung Cancer Cells in Tumorspheroids via the Intrinsic Pathway. Biomolecules, 10(11), 1521.

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Fas-induced Apoptosis Kinetics Assay

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Jurkat cells were seeded in 18 wells of 6-well plates (5×10⁵ cells/well) in RPMI medium with 5% FBS. The next day, the cells were washed once with phosphate buffered saline (PBS) and re-suspended in 1 mL of RPMI medium containing 12.5 ng/mL of anti-human Fas IgM (clone CH11). At each time point (0, 0.5, 1, 2, 4, and 8 hr, N = 3 for each time point), we harvested 500 µL of cell suspension, centrifuged it at 100 g for 5 min, transferred the medium to fresh microfuge tubes, and froze both the media and cell pellets separately at −80°C until the assays for LDH, n-DNA, fortilin, Cyt C, and fCK-18, were performed. For 7-AAD staining, we added 20 µL of 7-AAD solution (BD Pharmingen) to 400 µL of cell suspension and incubated it for 10 min at room temperature, shielded from light. We counted total and 7-AAD-positive cells under the fluorescence microscope as described previously [26 (link)]. The integrity of the plasma membrane of the cells with positive 7-AAD signal is compromised. At least 200 cells were counted and the 7-ADD index was calculated as (the number of 7-AAD-positive cells)/(the number of total cells)*100.

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