FITC-labelled anti-IA/IE, PE-labelled anti-Vβ11, anti-TNFα, anti-CD45.2, anti-CD40, APC-conjugated anti-CD80, anti-IFNγ, anti-CD69, PeCy7-labelled anti-CD11c, PercypCy5.5-labeled anti-CD44, anti-IL-2 and APC-Cy7-conjugated anti-CD62L were obtained from BD Pharmingen (San Diego, CA, USA). PE- labelled anti-CD86, PE-efluor 610-labelled anti-CD25 and Pacific blue conjugated-Annexin V were from Biolegend (San Diego, CA, USA). Alexa 405-conjugated anti-CD4 was obtained from CALTAG lab (Buckingham, UK). APC-conjugated anti-IA/IE and PE-labelled anti-CCR7 were obtained from eBioscience (San Diego, CA, USA). All stainings were performed in presence of mouse Fc Block (BD Biosciences). Dead cells were stained using the Live/Death detection kit with a near-infrared dye (Invitrogen). The samples were analysed using CyAn ADP Analyser (Beckman Coulter, Brea, CA, USA) and the data were analysed using FlowJo version 9.2 (Tree Star inc, Ashland, Oregon, USA).
Pacific blue conjugated annexin 5
Manufactured by BioLegend
Sourced in United States
Pacific Blue-conjugated Annexin V is a fluorescent-labeled protein that binds to phosphatidylserine on the surface of apoptotic cells. It can be used to detect and quantify apoptosis in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Annexin 5 binding buffer, by BioLegend (3 mentions)
Mouse fc block, by BD (1 mentions)
Anti cd45, by BD (1 mentions)
Live death detection kit, by Thermo Fisher Scientific (1 mentions)
Anti il 2, by BD (1 mentions)
Near infrared dye, by Thermo Fisher Scientific (1 mentions)
Cyan adp analyser, by Beckman Coulter (1 mentions)
Anti cd69, by BD (1 mentions)
Anti ifn γ, by BD (1 mentions)
Anti cd40, by BD (1 mentions)
Apc conjugated anti cd80, by BD (1 mentions)
Anti tnf α, by BD (1 mentions)
Macsquant vyb flow cytometer, by Miltenyi Biotec (1 mentions)
Pe conjugated streptavidin, by Jackson ImmunoResearch (1 mentions)
Tim 3 clone f38 2e2, by BioLegend (1 mentions)
Facscalibur, by BD (1 mentions)
Annexin 5 apoptosis detection kit, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
7 aad, by Thermo Fisher Scientific (1 mentions)
Propidium iodide solution, by Biotium (1 mentions)
7 protocols using pacific blue conjugated annexin 5
1
Multiparametric Flow Cytometry Analysis
2
Annexin-V Staining of Cell Suspensions
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Cell suspensions were prepared, counted and equal numbers of cells were allowed to rest for 2h in tissue culture medium at 37°C and 5% CO2. For analysis of Annexin-V staining without resting, aliquots of freshly isolated cells were dispensed into the 96-well round-bottom plates after preparation of single cell suspensions. Cells were then stained with the appropriate antibodies as detailed above and were washed once with flow cytometry buffer and once with Annexin-V binding buffer (Biolegend). Pacific Blue conjugated Annexin-V (Biolegend) staining was performed in Annexin-V binding buffer at room temperature for 15 min and washed and resuspended in Annexin-V binding buffer and kept at 4°C until analysis.
3
Overcoming PD-L1-mediated Gemcitabine Resistance
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B16F10 cells were co-cultured with fresh BM cells with or without 1 μM gemcitabine for 48 hours. To test the role of PD-L1 in cell response to the drug, B16F10 cells were pre-incubated with 2 μg/mL of PD-L1 blocking antibody (eBioscience, San Diego, CA) for 2 hours before co-culture with freshly isolated BM cells and treatment with gemcitabine (1 μM). To test cell viability, B16F10 cells were treated with 5 μM PH797804 during co-culture. To examine the role of p38 in the PD-L1 mediated drug resistance, B16F10 cells were treated with 5 μM PH797804 and 100 μM gemcitabine. After 48 hours of co-culture, single-cell suspensions were prepared with cold PBS buffer. After two washes, cells (1 × 106 cells/mL) were resuspended in 500 μL of annexin V binding buffer (Biolegend). Aliquots (100 μL) of the cell suspension were incubated with 5 μL of Pacific blue–conjugated annexin V (Biolegend) and 5 μL of propidium iodide (PI) solution (Biotium, Hayward, CA) for 15 minutes at room temperature in darkness. After staining, 400 μL of annexin binding buffer was added to the cells, which were immediately analyzed by flow cytometry.
4
T Cell Phenotyping and Viability Assay
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For surface marker staining, 1 × 105 T cells were seeded in 96-well plates with indicated target cells (unirradiated) at a 2:1 E:T ratio unless otherwise noted. Experiments with 2.5 × 105 T cells were performed in 24-well plates. When indicated, γ-irradiated (100 Gy) K562 targets were used. When indicated, CD28 monoclonal antibody (clone CD28.2; eBiosciences) was applied at 10 μg/mL to provide CD28 costimulatory signal. Cell mixtures were incubated at 37 °C, and analyzed at the indicated time points with fluorescently labeled monoclonal antibodies binding CCR7 (clone REA108), CD19 (clone LT19), CD25 (clone BC96), CD27 (clone M-T271), CD45RA (clone T6D11), CD57 (clone TB03), PD-1 (PD1.3.1.3), PD-L1 (clone 29E.2A3), and Tim-3 (clone F38-2E2) (BioLegend and Miltenyi Biotec). V500-conjugated Annexin V (BD Biosciences) and Pacific Blue-conjugated Annexin V (BioLegend) were used to detect pre-apoptotic cells. CAR expression was probed with Protein L (Genscript) followed by PE-conjugated streptavidin (Jackson Immunoresearch) or with APC-conjugated polyclonal antibody binding human IgG Fcγ (Jackson Immunoresearch). EGFRt expression was probed with biotinylated Erbitux followed by PE-conjugated streptavidin. Analyses were performed on a MACSQuant VYB flow cytometer (Miltenyi Biotec) equipped with 405-, 488-, and 561-nm lasers. Data were processed using FlowJo software (TreeStar).
5
Cell Proliferation and Apoptosis Assays
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Cell proliferation was evaluated by counting cell numbers every day or by performing WST assay (BioVision, Mountain View, CA). For cell apoptosis analysis, cells were stained with Pacific Blue-conjugated Annexin V (Biolegend, San Diego, CA) and propidium iodide (Annexin V apoptosis detection kit, BD Pharmingen, San Diego, CA) according to the manufacturer's instruction. The stained cells were analyzed immediately using a flow cytometer (FACS-Calibur; Becton-Dickinson).
6
Annexin V and 7-AAD Cell Apoptosis Assay
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Cultured cells were collected and stained with Pacific Blue‐conjugated annexin V (BioLegend) in binding buffer for annexin V (Thermo Fisher Scientific) at room temperature for 15 min. Stained cells were washed once with binding buffer and resuspended in binding buffer containing 7‐AAD (Thermo Fisher Scientific) before being analyzed on a FACSCanto II flow cytometer (BD Biosciences). All data were analyzed using FlowJo (BD Biosciences).
7
Antibody-induced Apoptosis in Cancer Cells
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To determine apoptosis of cancer cells due to antibody treatment, LN18 and GSC11 cells were treated with 2 μg/ml IgG or 86C for 24 hours. After 24 hours of treatment, single-cell suspensions were prepared with cold PBS buffer. After two washes, cells (1 × 106 cells/ml) were resuspended in 500 μl of Annexin V binding buffer (BioLegend, San Diego, CA). Aliquots (100 μl) of the cell suspension were incubated with 5 μl of Pacific blue–conjugated Annexin V (BioLegend) and 5 μl of propidium iodide solution (Biotium, Hayward, CA) for 15 minutes at room temperature in darkness. After staining, 400 μl of Annexin V–binding buffer was added to the cells, which were then immediately analyzed by flow cytometry.
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