Splenic DCs were rested 1 h, stimulated with different cytokines for 20 to 24 h, and apoptosis was analyzed by Annexin V and 7-AAD staining (BD Biosciences). For some experiments, the cells were incubated with 20 μM of serine protease inhibitor 3,4 dichloroisocoumarin (DCI) for 1 h before cytokine stimulation.
Annexin 5 and 7 aad staining
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Annexin V and 7-AAD are fluorescent dyes used in flow cytometry to identify and quantify apoptotic cells. Annexin V binds to phosphatidylserine, which is exposed on the cell surface during early apoptosis. 7-AAD is a DNA-binding dye that can enter and stain late apoptotic or necrotic cells. These stains are commonly used together to differentiate between viable, early apoptotic, and late apoptotic/necrotic cell populations.
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Lab products found in correlation
Facscalibur, by BD (2 mentions)
Fluo 4 am dye, by Thermo Fisher Scientific (1 mentions)
Facscanto, by BD (1 mentions)
Infinicyt software version 2, by Cytognos (1 mentions)
Lsrii analyzer, by BD (1 mentions)
Vybrant dyecycle violet stain, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm plus kit, by BD (1 mentions)
Alexa fluor 647 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Fluorescently conjugated antibodies, by BD (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Golgistop, by BD (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Brdu flow kit, by BD (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
Aria 3, by BD (1 mentions)
8 protocols using annexin 5 and 7 aad staining
1
Cytokine-Induced Splenic DC Apoptosis
2
Flow Cytometric Analysis of Apoptosis and Calcium Flux
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The procedure for the flow cytometric analysis was performed as described37 (link). The antibodies used in this study were as follows: anti-phycoerythrin-conjugated anti-mouse CD86 (clone GL1; BD PharMingen) and anti-phycoerythrin-Cy7 (PE-Cy7)-conjugated CD95 (clone Jo2, BD PharMingen). The cells were analysed using a FACSCanto (Becton Dickinson) with FACS Express 3.0 software. For detecting apoptotic cells, Annexin V and 7-AAD staining (BD PharMingen) were performed according to the manufacturer's protocols. The levels of calcium flux after anti-IgM stimulation were examined by labelling primary splenic B cells with Fluo-4AM dye (Invitrogen, 1 μM) at a density of 2 × 106 cells per ml in RPMI for 30 min at room temperature. The labelled cells were then washed and resuspended in Hank's balanced salt solution and stimulated by anti-IgM (25 or 10 μg ml−1) immediately before FACS analysis using a FACSCalibur (Becton Dickinson).
3
Cellular Immunophenotyping by Flow Cytometry
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After a washing in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) and 0.09% azide (AZ; wash solution, WS), cells were resuspended in 100 μL WS. 1 × 106 cells were incubated with an adequate concentration of antibodies (Table 4 ) for 15 min at RT in the dark. Samples were acquired on an FACSCanto II cytometer (Beckton Dickinson Biosciences, San Jose, CA, USA) equipped with FACSDiva software version 1.7 (BD Biosciences). Analyses were performed using Infinicyt software version 2.0 (Cytognos, Salamanca, Spain). Annexin V and 7-AAD staining (BD Bioscience, USA) were used to exclude nonviable cells. Unstained cells were used to assess background fluorescence. The results were expressed as the absolute cell count (dual-platform method). Experiments were performed in triplicate.
4
Cell Cycle and Apoptosis Analysis
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For the cell-cycle analysis, cells were collected, washed 3 times with 1× DPBS, fixed, and permeabilized with 75% ethanol at −20°C overnight. The cells were further incubated with 5 μM PI (Sigma) or Vybrant DyeCycle Violet stain (Thermo Fisher Scientific, V35003) for 30 min at 37°C, followed by the flow cytometry analysis using the BD LSRII analyzer. The percentage of sub-G1 was monitored using FlowJo software (v.10.8.1). Annexin V and 7-AAD staining (BD Biosciences, 559763) was performed according to the manufacturer’s instructions. Briefly, the cells were collected by centrifugation, resuspended in 500 μL 1× annexin V binding buffer, incubated with 5 μL annexin V and 5 μL 7-AAD at RT for 15 min in the dark, and followed by the flow cytometry analysis.
5
Multiparameter Flow Cytometric Analysis
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Single cell suspensions of thymus, lymph node (LN), and spleen were stained for cell surface proteins with fluorescently conjugated antibodies (BD Biosciences) and were analyzed on a FACSCalibur (BD Biosciences). Data were analyzed using FlowJo software (TreeStar Inc.). Data are presented gated on live cells as determined by FSC vs. SSC unless otherwise indicated. Annexin-V and 7-AAD staining was performed according to the manufacturer’s instructions (BD Biosciences). Intracellular cytokine and BIM staining were performed on cells that had been fixed and permeabilized using the BD Cytofix/Cytoperm Plus kit. For cytokine staining, cells were pretreated with PMA (Sigma, 50 ng/ml) and ionomycin (Sigma, 750 ng/ml) for a total of 6h and with GolgiStop (BD Biosciences) for the final 4h. Fluorescent anti-cytokine Abs were from BD Biosciences. Anti-BIM Ab was from Cell Signaling and secondary Alexa Fluor 647 goat anti-rabbit IgG was from Life Technologies.
6
Evaluating Proliferation and Apoptosis in T. cruzi Infection
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T. cruzi-infected WT and IL-17RA KO mice were given BrdU (1 mg/ml, Sigma-Aldrich)/1% sucrose in the drinking water (carefully protected from light) ad libitum. After 5 days, mice were sacrificed and spleen mononuclear cells were collected and stained with surface antibodies. Incorporated BrdU was detected with a BrdU Flow kit according to the manufacturer's specifications (BD Biosciences).
Apoptosis was determined by Annexin V and 7AAD staining according to the manufacturer's specifications (BD Biosciences). Mitochondrial depolarization (ψ) was measured by FACS using 50 nM TMRE (Invitrogen) as described (24 (link)). When indicated, 7AAD or LIVE/DEAD Fixable Aqua dead cell stain was used in combination to identify live, early apoptotic and late apoptotic/necrotic cells.
Apoptosis was determined by Annexin V and 7AAD staining according to the manufacturer's specifications (BD Biosciences). Mitochondrial depolarization (ψ) was measured by FACS using 50 nM TMRE (Invitrogen) as described (24 (link)). When indicated, 7AAD or LIVE/DEAD Fixable Aqua dead cell stain was used in combination to identify live, early apoptotic and late apoptotic/necrotic cells.
7
Cell Proliferation, Apoptosis, and Cycle Assays
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Human colon epithelial cell lines NCM 460 and FHC were used in this study. Cells were cultured in DMEM (Gibco) supplemented with 10% serum (Hyclone) for routine cultures and in DMEM with 0.5% serum for cell function experiments. To evaluate the cell proliferative potency, cells were seeded in a 96-well-plate at a density of 3000 cells/well and a Cell Counting Kit-8 (CCK-8, Dojindo) assay was performed. OD 450 values were measured every 24 h for 6 days. Cell apoptosis was induced by 50 ng/mL TNF-α (Peprotech) for 3 days. Annexin V and 7-AAD staining (BD bioscience) was performed, and cells were tested by a flow cytometer (Aria III, BD bioscience). Cell cycle analysis was measured by in vitro BrdU incorporation and performed following the manufacturer's instructions (MultiSciences). BrdU-incorporated cells were measured by flow cytometry. Data were analyzed by Flowjo 10.0 software.
8
NK Cell Characterization by Flow Cytometry
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For detection of surface markers, cell suspensions were stained with fluorescent antibodies for 30 min at 4 C. For measurement of IFN-g, perforin and granzyme B production, cells were stimulated with 20 ng/mL IL-15 and IL-12 for 12 h, then with brefeldin A at a final concentration of 10 mg/mL for 4 h. Intracellular staining was performed using fixation and permeabilization buffer in accordance with the manufacturer's guidelines. For detection of NK cell viability, NK cells were stained with Annexin V and 7-AAD staining according to the manufacturer's instructions (BD Biosciences). For assessment of lipids, NK cells were stained with Bodipy reagents and determined by flow cytometry.
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