Ear tissue was prepared as previously described.40 (link) Briefly, the two sheets of infected ear dermis were separated, deposited in DMEM containing 100 U/ml penicillin, 100 μg/ml streptomycin and 0.2 mg/ml Liberase CI purified enzyme blend (Roche Diagnostics Corp., Chicago, IL, USA) and incubated for 1 h and 30 min at 37 °C. Digested tissue was placed in a grinder and processed in a tissue homogenizer (Medimachine; Becton Dickenson, Chicago, IL, USA). To obtain neutrophils recruited to the site of infection in the skin, LYS-eGFP mice were inoculated in the ear dermis with 1–2 × 106Lm-RFP or Lm-NT-OVA-RFP or 1 × 106T. gondii-RFP parasites in 10 μl. After 12 h, ear tissue was prepared as described above and infected (eGFPhiRFP+) and uninfected (eGFPhiRFP−) neutrophils were sorted from dermal tissue using a FACSVantage or a FACsAria (BD Biosciences, Chicago, IL, USA) cell sorter. Sorted populations were washed once and immediately analyzed for apoptotic markers or cultured with mouse BM-DCs. Presorted neutrophil populations recruited to the site at 2 and 16 h after infection were stained with Annexin-V-APC and 7-AAD (BD Biosciences), and cells were analyzed by flow cytometry.
Annexin 5 apc and 7 aad
Manufactured by BD
Sourced in United States
Annexin-V-APC and 7-AAD are fluorescent dyes used in flow cytometry analysis. Annexin-V-APC binds to phosphatidylserine, a marker of apoptotic cells. 7-AAD is a DNA-binding dye that stains late-stage apoptotic and necrotic cells. These reagents are used to detect and quantify cell death and apoptosis.
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Lab products found in correlation
Medimachine, by BD (1 mentions)
Facsaria, by BD (1 mentions)
Facsvantage, by BD (1 mentions)
Liberase ci purified enzyme blend, by Roche (1 mentions)
Rnase a, by Fujifilm (1 mentions)
Epics altra, by Beckman Coulter (1 mentions)
7 aad, by BD (1 mentions)
Facscanto, by BD (1 mentions)
Cytoflex s, by Beckman Coulter (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Kaluza analysis software, by Beckman Coulter (1 mentions)
Isovolt titan x ray generator, by GE Healthcare (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Cytoflex s flow cytometer, by Beckman Coulter (1 mentions)
Facsverse, by BD (1 mentions)
Canto flow cytometer, by BD (1 mentions)
Annexin 5 binding buffer, by BD (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Facsdiva, by BD (1 mentions)
13 protocols using annexin 5 apc and 7 aad
1
Isolation and Analysis of Recruited Neutrophils
2
Cell Cycle and Apoptosis Analysis by Flow Cytometry
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The percentage of cells in different phases of the cell cycle, including the sub-G1 population, was measured by flow cytometry using propidium iodide (PI, Wako) staining46 . In brief, cells were treated with each compound for the indicated time, fixed with 70% ethanol at 4°C, and then stained with 50 μg/mL PI and 10 μg/mL RNase A (Wako) for 20 min at 37°C. PI fluorescence was measured on an EPICS ALTRA (Beckman Coulter, Brea, CA).
Detection of early apoptotic cells was determined by using annexin V/APC and 7AAD (BD Biosciences, San Jose, CA) according to the manufacturer's protocol. Briefly, 1 × 105 cells were exposed to each compound and washed with PBS twice. They were then incubated at room temperature with annexin V/APC and 7-AAD for 15 min. Annexin V/APC- and 7-AAD-stained cells were enumerated with a FACSCanto (BD Biosciences). Annexin V/APC-positive or -negative cells were regarded as apoptotic and non-apoptotic cells, respectively.
In the vector-transfection experiments, we co-transfected the EGFP expression vector as a transfectant marker with DN-TCF7L2 (Merck Millipore, Billerica, MA) or an active, mutated human β-catenin (S37A and S45A) expression vector (Daiichi Sankyo Co. Ltd.). EGFP-negative group were gated out by the cutoff results of non-transfectant during FCM analysis.
Detection of early apoptotic cells was determined by using annexin V/APC and 7AAD (BD Biosciences, San Jose, CA) according to the manufacturer's protocol. Briefly, 1 × 105 cells were exposed to each compound and washed with PBS twice. They were then incubated at room temperature with annexin V/APC and 7-AAD for 15 min. Annexin V/APC- and 7-AAD-stained cells were enumerated with a FACSCanto (BD Biosciences). Annexin V/APC-positive or -negative cells were regarded as apoptotic and non-apoptotic cells, respectively.
In the vector-transfection experiments, we co-transfected the EGFP expression vector as a transfectant marker with DN-TCF7L2 (Merck Millipore, Billerica, MA) or an active, mutated human β-catenin (S37A and S45A) expression vector (Daiichi Sankyo Co. Ltd.). EGFP-negative group were gated out by the cutoff results of non-transfectant during FCM analysis.
3
Evaluating Senescence in Cell Treatments
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To investigate evolving senescence during KI or irradiation treatment, we seeded cells at low confluence in cell culture flasks. After 24 h of settlement, cells were treated with either kinase inhibitor, irradiation, or combination of both. As a control, cells were treated with DMSO only. On day 6 after treatment, medium was exchanged including DMSO or kinase inhibitor again. After 10 days of treatment, cells were collected and stained as previously published (23 (link)). Briefly, cells were treated with 100 nM Bafilomycin A1 (Merck, Darmstadt, Germany) for 30 min (37°C). Afterwards, Hoechst dye was added for another 30 min (37°C) and finally cells were treated with C12-FDG for 60 min (37°C). After centrifugation cells were resuspended in Ringer solution and stained with Annexin V-APC and 7-AAD (BD, Heidelberg, Germany) for 30 min on ice. Finally, C12-FDG positive cells were measured using flow cytometry (Cytoflex S, Beckman Coulter, Brea, CA, USA).
4
Apoptosis and Necrosis Assessment
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Adequate count of cells was seeded into T25-flasks to reach a confluence of around 50 % and settled down over night. On the next day medium was exchanged with cell culture medium containing a reduced amount of 2 % FBS to avoid bias in outcome because of cells triggered strongly for proliferation. Additionally, kinase inhibitor was added in a certain concentration and after 3 h of incubation cells were irradiated with a dose of 2 Gy by an ISOVOLT Titan X-ray generator (GE, Ahrensburg, Germany). After 48 h of incubation cells and supernatant were collected and stained using Annexin-V-APC and 7-AAD (both: BD Biosciences, Franklin Lakes, NJ, USA) for 30 min on ice. Cells were analyzed using a Beckman coulter cytometer (Cytoflex, Beckman Coulter, Brea, CA, USA). Cells neither positive for Annexin-V nor 7-AAD were defined as “alive”, Annexin-V-positive and 7-AAD-negativ cells as “apoptotic” and double-positive cells as “necrotic. For data evaluation, the Kaluza analysis software (Beckman Coulter, Brea, CA, USA) was used.
5
Apoptosis Measurement by Flow Cytometry
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The Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, CA, United States) was used to perform flow cytometry to measure the apoptotic rate. Briefly, after harvest, the floating and attached cells were washed with cold PBS, resuspended in Annexin V binding buffer, stained with Annexin V-FITC and PI, and then analyzed by flow cytometry. The flow cytometer (BD Biosciences, CA, United States) was used to measure 1 × 106 cells for each experiment, and Flow Jo v10.2 (BD Biosciences, CA, United States) was used for analysis. The impact of lncRNA-HRAT on apoptosis triggered by H/R was tested using Annexin V-APC and 7-AAD (BD Biosciences, CA, United States). The methods used were similar to those used for the Annexin V-FITC Apoptosis Detection Kit.
6
Apoptosis Profiling of Transduced Cell Lines
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The percentages of apoptotic cells were determined in SeAx and HH cells harvested on day 8 after transduction with the lentiviral miRZIP vectors aiming at an infection percentage of >95%. Briefly, cells were washed twice with cold phosphate-buffered saline and resuspended at a concentration of 1 × 106 cells/mL in 1X Binding Buffer. Cells were stained with Annexin V APC and 7AAD according to the manufacturer’s protocol (BD Biosciences, San Jose, CA, USA) and analyzed via flow cytometry (CytoFLEX S Flow Cytometer, Beckman Coulter, Indianapolis, IN, USA).
7
Apoptosis Induction in Cells
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Cells were pretreated with nystatin (25 µg/mL), mβCD (10 mM), or CPZ (5 µM) for 30 min before treatment with 100 µg/mL of CTX. Cells were harvested and washed twice with PBS. Equal cell numbers were resuspended in tubes. Cells were stained with Annexin V-APC and 7-AAD (BD Biosciences). FACSVerse (BD Biosciences) was used to measure apoptosis.
8
Evaluating Myeloma Cell Survival
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Myeloma cells (1 × 105/well) were seeded in triplicates in 96-wells and incubated as indicated. In coculture experiments, myeloma cells were seeded onto 50.000 HS-5 stroma cells, i.e. in a ratio of 2:1. Survival of myeloma cells was evaluated by flow cytometry utilizing Annexin-V-APC and 7-AAD staining (BD Biosciences, USA) of gated myeloma cells (CD38high/CD45 low-neg).
9
Apoptosis Assay Using Flow Cytometry
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Cells were seeded in 24‐well plates (50,000 cells per well). Drugs were added 24 hours postseeding and incubated for the indicated periods. Cells were detached and resuspended in Annexin‐V‐binding buffer (BD Pharmingen) and stained with Annexin‐V‐APC and 7‐AAD (BD Pharmingen). The samples were subsequently analyzed with a BD Canto flow cytometer.
10
Apoptosis Analysis of MPC5 Cells
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MPC5 cells were collected in phosphate-buffered saline (PBS), washed with a fluorescence-activated cell sorter (FACS) buffer and stained with annexin V-APC and 7-AAD (BD Biosciences, San Jose, CA, USA) in the dark for 30 min at 4°C. Subsequently, cells were washed once with FACS buffer and were resuspended. Then, 104 cells were obtained using a FACScan Flow Cytometer (BD Biosciences, San Jose, CA, USA). The analysis was performed using FlowJo Software 7.6 (TreeStar, Ashland, OR, USA). Apoptotic cells were expressed as a percentage of the total cells.
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