HD11 cells were harvested 48 h after stimulation using 200 µL PBS + 5 mM UltraPure EDTA and centrifuged at 400× g. The cells were transferred to a 96-well V-bottom plate, washed in PBS with 0.5% bovine serum albumin, 0.005% sodium azide (FACS buffer; both from Sigma-Aldrich), and stained for viability in 200 µL FACS buffer with 1.25 µg/mL 7-aminoactinomycin D (7-AAD; BD Biosciences, Pharmingen, San Diego, CA, USA). The staining of the cells was analyzed using the 488 nm laser of a CytoFLEX LX flow cytometer (Beckman Coulter Inc., Brea, CA, USA). The fraction of viable 7-AAD-negative HD11 cells was determined using FlowJo Software v. 10.5 (FlowJo LCC, Ashland, OR, USA). The cytotoxicity of the extracted antigens and purified bacteria from the octavalent vaccine was expressed as the concentration at which 50% of HD11 cells had died, i.e., the 50% lethal concentration (LC50).
7 aad
Manufactured by FlowJo
Sourced in United States
7-AAD (7-Aminoactinomycin D) is a fluorescent dye used in flow cytometry to detect DNA content and cell viability. It binds to DNA and emits a strong fluorescent signal, allowing for the identification of dead or dying cells in a sample.
Automatically generated - may contain errors
Lab products found in correlation
7 aminoactinomycin d 7 aad, by BD (1 mentions)
Cytoflex lx flow cytometer, by Beckman Coulter (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Facs buffer, by Merck Group (1 mentions)
Annexin 5 fitc kit, by Abcam (1 mentions)
Flowjo v 10, by FlowJo (1 mentions)
7 aad, by Abcam (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
7 aad, by Merck Group (1 mentions)
Fitc conjugated anti brdu antibody b44, by BD (1 mentions)
Goat anti mouse igg conjugated with alexafluor 488, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg conjugated with alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Facscalibur flow cytometer, by FlowJo (1 mentions)
Mildform 10n, by Fujifilm (1 mentions)
Saponin, by Merck Group (1 mentions)
7 aad, by Thermo Fisher Scientific (1 mentions)
Saponin, by Fujifilm (1 mentions)
Bovine serum albumin fraction 5, by Merck Group (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
5 protocols using 7 aad
1
Viability Assay for Vaccine Cytotoxicity
2
Cell Cycle and Apoptosis Analysis
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For cell cycle analysis, scramble siRNA-, and siRNA-transfected cells were washed with PBS and incubated in EtOH 80% for 30 min. After centrifugation and washing steps, cells were resuspended in DAPI/TX-100 solution (9 mL PBS; 1 mL Triton X-100; 10 μL DAPI 1 μg/mL) and incubated for 30 min at room temperature. Cell cycle analysis was next performed by FlowJo v.10.6.1 software, which provides a simple interface to perform sophisticated univariate DNA/Cell Cycle analysis using the Dean-Jett-Fox model71 .
Quantification of apoptotic cells was performed using the Annexin V-FITC Kit (Abcam, cat n° ab14082) and 7-AAD (cat n° 559925), for the exclusion of nonviable cells and detectable in far red, following the manufacturer’s instructions. Briefly, 1 × 105 cells were incubated with 5 µl of both Annexin V and 7-AAD in the proper buffer for 5 min in the dark and then acquired by FACS and analyzed by using FlowJo v.10.6.1.
Quantification of apoptotic cells was performed using the Annexin V-FITC Kit (Abcam, cat n° ab14082) and 7-AAD (cat n° 559925), for the exclusion of nonviable cells and detectable in far red, following the manufacturer’s instructions. Briefly, 1 × 105 cells were incubated with 5 µl of both Annexin V and 7-AAD in the proper buffer for 5 min in the dark and then acquired by FACS and analyzed by using FlowJo v.10.6.1.
3
Cell Proliferation Analysis by BrdU Incorporation
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Cells were grown in 10 cm dishes in 10% CSS for 3 days before the media was changed to 1.5% CSS and ATRA and/or EPI-002 were added at the indicated concentrations. After treatments, the cells were labeled with BrdU (10 μM) for 2 h and then trypsinized, washed, and fixed in 70% EtOH/PBS at −20 °C. Approximately 2.5–5.0 × 105 of fixed cells were labeled with fluorescein (FITC)-conjugated anti-BrdU antibody (B44; BD Biosciences) and the DNA content was stained with 7-AAD (Sigma). The data were collected with a BD FACSCalibur flow cytometer (BD Biosciences). FITC and 7-AAD fluorescence were detected in the FL1 and FL3 channels, respectively, using CellQuest Pro and analyzed by FlowJo V10 software (Ashland, Oregon). Pre-gating was performed on all samples by plotting FL3-W × FL3-H to exclude doublets from the analysis.
4
EBV Receptor and Cytoskeleton Expression
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To determine the efficiency of eGFP‐EBV infection, cells were detached using trypsin and suspended in FACS solution (2% FBS in PBS (−)) containing 7‐AAD (eBioscience) to distinguish the dead cells. To determine EBV receptor expression, cells were exposed to H. pylori (MOI 100) for 7 h under cell culture conditions. Cells were fixed with Mildform 10 N (FUJIFILM Wako Pure Chemical Corp.) for 10 min at 25℃. After washing with PBS (−), the cells were incubated with PBS (−) containing 0.5% saponin (Wako) for 20 min at 25℃. Cells were incubated with PBS (−) containing 0.5% saponin and 5% bovine serum albumin fraction V (Sigma‐Aldrich) for 1 h at 25℃. The cells were stained with rabbit anti‐human‐EphA2 antibody (1C1, Novus Biological) or with mouse anti‐human NMHC‐IIA (GT218; Genetex) for 30 min on ice. Cells were then washed with FACS solution and stained with goat anti‐rabbit IgG conjugated with Alexa Fluor 488 or goat anti‐mouse IgG conjugated with Alexa Fluor 488 (Thermo Fisher Scientific) for 30 min on ice. Finally, cells were suspended in FACS solution containing 7‐AAD and analyzed using a FACS Calibur flow cytometer and FlowJo software (FlowJo LLC).
5
Apoptosis Analysis of Prostate Cancer Cells
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LNCaP, Enza-R and Bica-R cells (1×106 cells/well) were plated in 6-well plates and cultured to 60% confluence for apoptosis analysis. Cells were irradiated at room temperature as described above. After 24 h, cells were washed with PBS and detached with 2.5 mM EDTA in PBS. Annexin V and 7-amino-actinomycin D (7-AAD; both from Sungene Biotech Co., Ltd) staining was performed according to the manufacturer's instructions. Staining was quantified using flow cytometry (CytoFLEX; Beckman Coulter, Inc.) by analyzing Annexin V-fluorescein isothiocyanate (FITC) and 7-AAD fluorescence using phycoerythrin emission signal detectors and FlowJo V10 software (FlowJo LLC).
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