Cultured cells were seeded at a density of 1 × 105 cells/well using a 6-well plate, and then vMyx-mLIGHT-FLuc/tdTr (MOI = 5) was added to the cells. After 24 h and 48 h, the cells were collected and washed twice with PBS¯ and staining buffer. Then, the cells were stained with anti-Annexin V antibody and 7-aminoactinomycin D (BioLegend, San Diego, CA, USA; kit # 640922) and analyzed for apoptosis/necrosis using flow cytometry (BD FACS Canto II). Annexin V was detected using an FITC channel and 7-AAD using a PerCP-Cy5.5 channel, and a region for live cells was defined. Non-infected cells were used as a control.
7 aminoactinomycin d
Manufactured by BioLegend
Sourced in United States, Germany
7-aminoactinomycin D is a fluorescent dye used in flow cytometry and other applications to distinguish viable cells from dead or dying cells. It binds to DNA and emits fluorescence when excited by a laser.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (6 mentions)
Annexin 5, by BioLegend (4 mentions)
Facscalibur, by BD (4 mentions)
Lsrfortessa, by BD (4 mentions)
Macsquant, by Miltenyi Biotec (3 mentions)
Facscanto 2 device, by BD (3 mentions)
Cytofix cytoperm kit, by BD (3 mentions)
Facsaria 2, by BD (3 mentions)
Annexin 5 fitc, by BioLegend (3 mentions)
Attune nxt, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (2 mentions)
7 aad, by BioLegend (2 mentions)
Canto 2, by BD (2 mentions)
Cytoflex flow cytometer, by Beckman Coulter (2 mentions)
Facsverse, by BD (2 mentions)
7 aminoactinomycin d, by BD (2 mentions)
Anti cd11b, by BioLegend (2 mentions)
Lsrfortessa x 20, by BD (2 mentions)
Fitc brdu flow kit, by BD (2 mentions)
72 protocols using 7 aminoactinomycin d
1
Apoptosis and Necrosis Analysis
2
Identification of M1 Macrophages and Regulatory T Cells
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Cells in the SVFs were suspended in the staining buffer (PBS containing 0.5% BSA and 2 Mm ethylenediaminetetraacetic acid) and then incubated with CD11b (BioLegend, clone: M1/70) and CD11c (BioLegend, clone: N418) antibodies or the control isotypes at 4 °C. Thirty minutes later, cells were then washed twice and resuspended in the staining buffer. After incubation with 7-amino-actinomycin D (BioLegend), the cells were analyzed by Attune NxT Flow Cytometer (ThermoFisher). The data analysis was performed by using FlowJo (Tree Star, Ashland, OR). Cells with double positive expression of CD11b and CD11c were identified as M1 macrophages. For intracellular FoxP3 staining, PE/Cyanine7 anti-mouse CD4 (BioLegend, # 100,528)-stained cells were resuspended in 1 ml of the True-Nuclear™ 1X Fix Concentrate to each tube, vortex and incubate at room temperature in the dark for 60 min, followed by adding 2 mL of the True-Nuclear™ 1X Perm Buffer (BioLegend, # 424,401) to each tube and then centrifuge tubes at 2100 rpm at room temperature for 8 min, and discard the supernatant. Tubes were decanted, blotted, washed twice with True-Nuclear™ 1X Perm Buffer. Resuspend the cell pellet in 100 µl of the True-Nuclear™ 1X Perm Buffer. For detection of intracellular antigen, add 1 μl of PE–anti-mouse FoxP3 antibody (BioLegend, # 126,404) to each tube and incubate in the dark at room temperature for 60 min.
3
Human Fetal Thymus Single-Cell Analysis
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Human fetal thymi, obtained from terminations of pregnancy at 14 and 17 post-conception weeks, were enzymatically dissociated using Liberase (Roche) and DNaseI (Roche). The resultant cell suspension was stained with the following antibodies directed against cell surface antigens for 30 min at 4°C: CD45::BV421 (BioLegend, H130) and HLA-DR::PE-Cy7 (BioLegend, L243); 7-aminoactinomycin D (BioLegend) was used as a viability marker. Live CD45− MHCII (major histocompatibility complex class II) intermediate-high cells were sorted in 250,000 cell aliquots using FACSAria III (BD Biosciences). Samples were then processed using the 10x Genomics Multiomics ATAC (Assay for Transposase-Accessible Chromatin using sequencing) + Gene Expression Kit according to the manufacturer’s protocol with some adaptations. Specifically, nuclei were isolated using a 0.1× diluted nuclei extraction buffer for 6 min before being captured into droplets on the 10x Genomics Chromium platform and sequenced on an Illumina NovaSeq machine. This study of human thymic tissue has been granted ethical approval and is publicly listed (IRAS ID 156910, CPMS ID 19587).
4
Apoptosis and Cell Death Assay
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Cell death was assessed by double staining of cells with FITC-conjugated annexin V (MEBCYTO© 4700 Apoptosis Kit; MBL©) and 7-Aminoactinomycin D (7-AAD; BioLegend©) as per manufacturer’s instructions.
5
OVA-Specific CD8+ T Cell Response
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C57BL/6J mice were treated with OVA (80 µg/mouse) without or with K-type CpG ODNs (8 or 40 µg CpG ODNs/mouse) or CA-CpG (8 µg CpG ODNs/mouse) into the ear pinna. Draining lymph nodes were collected at day 7, and lymph node cells were prepared for determination of OVA257–264-specific CD8+ CTL responses. Lymph node cells (1 × 106 cells) were added to the wells of a 96 half-well plate and then stimulated with OVA257–264 peptide (SIINFEKL, final concentration 5 µg/mL) for 24 h at 37°C. After the incubation, the concentration of IFN-γ in the supernatants was analyzed by means of ELISA. For the tetramer assay, lymph node cells (1 × 106 cells) were stained with purified anti-mouse CD16/CD32 antibody and then with the phycoerythrin-labeled H-2Kb/OVA257–264 tetramer (MBL, Aichi, Japan). Fluorescein isothiocyanate-conjugated CD8α (Abcam, Cambridge, UK), allophycocyanin-conjugated CD44 (BioLegend), and 7-amino-actinomycin D (BioLegend) were then added. The number of CD8α+ CD44+ tetramer+ cells was determined by flow cytometry.
6
Evaluating MM Cell Viability with sEVs
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In the absence or presence of bortezomib (7.5 nM for MM1S, RPMI8226, and U266 cells, 30 nM for H929 cells), MM cells (3×105 cells/mL) were treated with or without BMSC-derived sEVs for 48 h and the viability was measured with the Cell Titer glo Luminescent Viability assay (Promega, Madison, WI, USA). MM cells were treated with or without endocytosis inhibitors for 4.5 h and the cell viability was determined using the Cell Titer glo Luminescent Viability assay. All viability assays were performed in triplicate and repeated in three independent experiments. For apoptosis analysis, the treated cells were stained with Annexin V-FITC (Biolegend, San Diego, CA, USA) and 7-Aminoactinomycin D (7-AAD, Biolegend) and apoptotic cells were determined using a CytoFLEX S flow cytometer (Beckman Coulter, Brea, CA, USA).
7
Evaluating CD38+ NK Cell-Mediated Treg Cytotoxicity
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CD38+ NK cells and autologous Treg cells were isolated from the peripheral blood of RA patients using flow cytometry-based methods, as described above. CD38+ NK cells were incubated in 50 μM C3G or an equal volume of PBS at 37 °C for 24 h. Isolated Treg cells were labeled with 5 μM carboxy fluorescein diacetate succinimidyl ester (CFSE; BioLegend) for 10 min at 37 °C. The pretreated CD38+ NK cells and Treg cells were washed and seeded at an effector-to-target cell (E:T) ratio of 20:1 in a 96-well U-bottom plate for 4 h at 37 °C. 7-Aminoactinomycin D (7-AAD; 5 μg/ml; BioLegend) was added to the culture, and the mixture was incubated for 20 min at 4 °C. Cell death was measured using flow cytometry. The percentage of specific lysis was calculated based on the following formula: 100 × (CFSE+ 7-AAD+ Treg cells/total number of CFSE+ Treg cells). The flow cytometry-based cytotoxicity assay for CD38+ NK cells against Treg cells was designed following a previously reported method [37 (link), 38 (link)].
8
Adipose Stromal Vascular Fraction Isolation
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Perigonadal VATs were minced and digested with type II collagenase (Sigma). Stromal vascular fraction (SVF) cells were obtained as described by Anderson et al. (18 (link)). After blocking with anti-CD16/32 antibodies (BD Biosciences, San Jose, CA), cells were stained with fluorophore-conjugated antibodies. Allophycocyanin (APC)-Cy7-B220, APC-TCRβ, phycoerythrin (PE)-Cy7-CD8, PerCP-Cy5.5-CD44, Alexa Fluor 488–CD62L, PE-Cy7-CD45.2, BV421-F4/80, APC-Cy7-CD11c, fluorescein isothiocyanate (FITC)-CD11b, PE–Siglec F, APC-CD11c, Pacific Blue–CD3, Pacific Blue–CD8ɑ, Pacific Blue–CD19, FITC-FCεIɑ, FITC–Pan NK, APC-Cy7-CD25, PerCP-Cy5-CD127, PE-IL33R, APC-CD11b, Alexa Fluor 488–CD45.1, and PerCP-Cy5.5-CD86 antibodies were from BioLegend (San Diego, CA). FITC-CD4 and eFluor 605-CD4 antibodies were from BD Biosciences and eBioscience (San Diego, CA), respectively. DAPI (Invitrogen, Carlsbad, CA) or 7-aminoactinomycin D (BioLegend) was added as viability dye. Flow cytometry experiments were performed on the BD LSR II and analyzed using FlowJo software (Tree Star, Ashland, OR).
9
Interrogating ZNF365 in Lung Cell Response
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A549 cells previously silenced for ZNF365 (24 h before) were seeded at 70% confluency and stimulated with 30 mU/mL of bleomycin (Cayman) in serum-free medium for 48 h. Normal human lung fibroblasts were seeded at 70% confluency, previously silenced (48 h before) for ZNF365, and stimulated with [0.1 and 0.5 μM] for 1 h. Cells were harvested and stained with PE Annexin V (556421, BD Biosciences, San Jose, CA, USA) and 7-aminoactinomycin D (7AAD, 559925, Biolegend, San Diego, CA, USA). Cells were analyzed by flow cytometry in a FACSAria flow cytometer (BD Biosciences). Results were analyzed with FlowJo 7.8 software (FlowJo, Ashland, OR, USA, Becton Dickinson, and Company).
10
Flow Cytometry Analysis of Virus-Infected Cells
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Flow cytometry was performed using direct fluorescence-conjugates and their respective controls. Phycoerythrin (PE)-labeled anti-human CD46 antibody and PE-labeled IgG1 isotype control were from eBioscience. CD117-PeCy7 and CD33-BV421 conjugates were from BD Biosciences. Dead cells were excluded using either 7-aminoactinomycin D (BioLegend) or using Fixable Aqua (Invitrogen; Thermo Fisher Scientific) following extracellular staining according to the manufacturer's instructions. Cells employed in the virus infection experiments were fixed using cytofix buffer (BD Biosciences). Acquisition was done using a Canto-II (BD Biosciences) or an Attune NxT (Life Technologies) cytometer. Viable, single cells were used for further analysis as depicted in Fig. S1 apart from tetramethylrhodamine ethyl ester (TMRE) staining where only subcellular debris was excluded.
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