For determination of apoptotic and necrotic cells, an Annexin V/Zombie assay (BioLegend) was used. Heparinized human whole blood samples were incubated with 6-FAM-labeled PFOB-NE at a dilution of 1:20 at 37 °C under constant shaking for 4 h. 200 µL of whole blood was taken, placed on ice and lysed 3 times for 15 min. Afterwards, leukocytes were re-suspended in PBS and incubated with Zombie Aqua Fixable Dye (BioLegend, 1:100) for 15 min. Cells were then washed with FACS-buffer (2% FCS, 2 mmol/L EDTA in PBS), re-suspended in Annexin V binding buffer and stained with APC-labeled Annexin V (BioLegend, 1:20) for 15 min. Flow cytometry was performed as described above. In our gating strategy neutrophils, monocytes or lymphocytes were located in the FSC and SSC, cell duplicates and dead cells were excluded and necrotic, apoptotic and living cells were discriminated by Annexin V and Zombie staining. A representative example of the gating strategy is shown in supplementary Figure S4 .
Apc labeled annexin 5
Manufactured by BioLegend
Sourced in United States
APC-labeled annexin V is a fluorescently-labeled protein that binds to phosphatidylserine, a molecule exposed on the surface of apoptotic cells. This product can be used to detect and quantify apoptosis in cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 binding buffer, by BioLegend (2 mentions)
Annexin 5, by BioLegend (2 mentions)
Apc labeled anti brdu antibody, by BioLegend (2 mentions)
Zombie aqua fixable dye, by BioLegend (1 mentions)
Cellquest pro, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Cba mouse inflammation kit, by BD (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Facsaria 3 flow cytometer, by BD (1 mentions)
24 well plate, by Corning (1 mentions)
7 protocols using apc labeled annexin 5
1
Annexin V/Zombie Assay for Apoptosis and Necrosis
2
Quantification of Apoptotic Cells by Flow Cytometry
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Flow cytometry was used for the quantitation of apoptotic cells by the co-staining of annexin V and propidium iodide (PI). Briefly, after 2 × 106 cells/well of a 96-well culture plate were treated with OA (1 mM) and/or antioxidant (10 μM) for 24 h, the cells were harvested for APC-labeled annexin V and PI staining and subjected to fluorescence-activated cell sorting (FACS) analysis. The harvested cells were incubated with 100 μL FACS staining buffer (1% BSA and 0.1% sodium azide) containing 5 μL APC-labeled annexin V (BioLegend, San Diego, CA, USA) and 2 μL PI (Sigma, St. Louis, MO, USA) for 15 min at RT in the dark. The stained samples were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). Data acquisition and analyses were performed using “Cell-Quest pro” (Becton Dickinson), and further data analyses for forward/side scatter gates were performed using FlowJo software (Tree Star, Inc., Ashland, OR, USA). Data of apoptosis (%) were displayed as the percentage of cell numbers stained with annexin V alone (Q3-plot) and dual staining annexin V + PI (Q2-plot) (n = 4).
3
Extracellular Vesicle Immunophenotyping
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One milliliter of EV suspension was stained with 1 μl of rabbit anti-mouse Rab7 antibody (clone ab137029, Abcam, San Francisco, CA) at 4 ºC overnight. The EVs were washed by ultracentrifugation at 160,000 xg for 90 min in an Optima MAX ultracentrifuge (Beckman Coulter) with a TLS55 rotor; 200 μl of suspension were collected from the bottom of the tube and stained with 1 μl of FITC-labeled anti-rabbit IgG antibody (clone 31,635, Thermo Fisher Scientific, Waltham, MA) at room temperature for 1 h. 800 μl of Hanks’ balanced salt solution (Gibco) were added and the EVs were washed again. 200 μl of suspension were collected from the bottom of the tube. Ten min before analysis, 1 μl of APC-labeled annexin V (BioLegend, San Diego, CA) and 200 μl of annexin V binding buffer (BioLegend) were added (annexin V is a protein that binds to phosphatidylserine when this phospholipid is present in the outer membrane layer). The EVs were analyzed in a FACSCalibur (Beckton Dickinson, San Jose, CA) flow cytometer previously washed with 1 L of 0.45 µm-filtered 0.5% sodium hypochlorite and 5 L of 0.45 µm-filtered distilled water. At least 900,000 events were acquired for each sample. Data were analyzed with FlowJo 10.4.2 software (FlowJo LLC, Ashland, OR).
4
Apoptosis Analysis of ATO-Treated NB4 Cells
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NB4 and NB4-R2 cells were seeded in 24-well plates and treated with ATO (1 µM) and vehicle (NaOH) for 24 h. Cells were then washed twice with ice cold PBS and resuspended in binding buffer containing 1 µg/ml propoidium iodide (PI) and 1 µg/ml APC-labeled annexin V (BioLegend, San Diego, CA, USA). All specimens were acquired by flow cytometry (FACSCalibur; Becton-Dickison) after incubation for 15 min at room temperature and analyzed using FlowJo software (Treestar, Inc., San Carlos, CA, USA).
5
Apoptosis and Cell Cycle Analysis
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To detect apoptosis, cells were washed with PBS and resuspended with Annexin V binding buffer at 1 × 106 cells/ml. 100ul cell suspension was stained with 5 ul APC-labeled Annexin V (BioLegend, Cat No. 640932) at room temperature for 1 h, and 10ul propidium iodide (PI) solution was added 10 min before flow cytometric analysis. Bromodeoxyuridine (BrdU, MedChemExpress, Cat No. HY-D0184) and PI staining was performed to detect the cell cycle distribution. Cells (1 × 106) were plated in 100 mm dishes with 10 μM BrdU for 1 h before harvesting. For detection of the cell cycle distribution in the T-ALL model, KO and WT mice received an initial intraperitoneal injection of BrdU (1 mg/6 g mouse weight) and were then maintained on 1.0 mg/ml BrdU in the drinking water for 24 h prior to sacrifice. Cells treated with BrdU were fixed in 75% ethanol at − 20 °C for at least 2 h and processed according to manufacturer’s instructions. An APC-labeled anti-BrdU antibody (BioLegend, Cat No. 364114) was used to detect BrdU, and a PI solution was added 10 min before flow cytometric analysis.
6
Mycobacterium tuberculosis Infection Modulates
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J774A.1 cells were cultured to near 100% confluence in 24-well plates (Corning) and infected with M. tuberculosis H37Rv at MOI =10 for 3 h. Cells were washed, and extracellular mycobacteria were eliminated with 10 µg/ml amikacin (Bruluart, Tultitlán, Mexico). After 1 h, the cells were washed and incubated in DMEM with 7% of EV-reduced fetal calf serum, 200 μl of annexin V binding buffer (BioLegend) and 2.5 μg (the protein concentration was determined by absorbance at 230nm) of EVs or annexin V-blocked EVs. [These EVs were incubated with 1 μl of unlabeled annexin V (BioLegend) for 15 min before use; the blocking of phosphatidylserine was confirmed by adding 1 μl of APC-labeled annexin V (BioLegend) and analyzing by flow cytometry]. After 44 h, the culture supernatants were collected and stored at −70°C, and CFU were determined by lysing the cells with 500 μl of 0.1% Triton X-100 (Sigma-Aldrich) for 7 min and performing serial dilutions in 7H9 broth; 10 μl of each dilution were plated on Middlebrook 7H10 agar with OADC growth supplement. Colonies were counted at days 7 and 15. The culture supernatants were analyzed with a CBA Mouse Inflammation Kit (BD Biosciences, San Jose, CA) to determine the concentrations of IL-6, IL-10, IL-12p70, MCP-1 and TNF-α; the samples were acquired in a FACSAria III flow cytometer (BD Biosciences). Data were analyzed with FlowJo 10.4.2 software.
7
Apoptosis and Cell Cycle Analysis
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To detect apoptosis, cells were washed with PBS and resuspended with Annexin V binding buffer at 1x10 6 cells/ml. 100ul cell suspension was stained with 5 ul APC-labeled Annexin V (BioLegend, Cat No. 640932) at room temperature for 1 h, and 10ul propidium iodide (PI) solution was added 10 min before ow cytometric analysis. Bromodeoxyuridine (BrdU, MedChemExpress, Cat No. HY-D0184) and PI staining was performed to detect the cell cycle distribution. Cells (1x10 6 ) were plated in 100 mm dishes with 10 µM BrdU for 1 h before harvesting. For detection of the cell cycle distribution in the T-ALL model, KO and WT mice received an initial intraperitoneal injection of BrdU (1 mg/6 g mouse weight) and were then maintained on 1.0 mg/ml BrdU in the drinking water for 24 h prior to sacri ce. Cells treated with BrdU were xed in 75% ethanol at -20°C for at least 2 h and processed according to manufacturer's instructions. An APC-labeled anti-BrdU antibody (BioLegend, Cat No. 364114) was used to detect BrdU, and a PI solution was added 10 min before ow cytometric analysis.
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