A total of 4 × 105 stimulated OVA-specific CD8+ T cells were added to 1 × 105 B16F10-mCherry-OVA and treated with fresh condition medium (1:2 dilution). After 48 h, cells were stained using anti-Annexin V, 7-AAD (BioLegend, 640930) and anti-CD8 for 20 min at 4 °C, and were immunophenotyped by flow cytometry using an LSRFortessa or a FACSCanto II (Becton Dickinson). For CGRP, 4 × 105 stimulated OVA-specific CD8+ T cells were added to 1 × 105 B16F10- mCherry-OVA and treated with CGRP (100 nM). After 24 h, the cells were stained using anti-Annexin V, 7-AAD (BioLegend, 640930) and anti-CD8 for 20 min at 4 °C, and were immunophenotyped by flow cytometry using an LSRFortessa or a FACSCanto II (Becton Dickinson). Cytokine expression levels were analysed after in vitro stimulation (PMA–ionomycin; see ‘Intracellular cytokine staining’).
Anti annexin 5
Manufactured by BioLegend
Sourced in United States
Anti-Annexin V is a laboratory reagent used in the detection and quantification of Annexin V, a protein involved in various cellular processes. It serves as a tool for researchers studying Annexin V-related phenomena.
Automatically generated - may contain errors
Lab products found in correlation
7 aad, by BioLegend (2 mentions)
Annexin 5 binding buffer, by BioLegend (2 mentions)
Facscanto 2, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Trustain fcx anti mouse cd16 32, by BioLegend (1 mentions)
Anti sca 1 d7, by BioLegend (1 mentions)
Pe anti mcd47 miap301, by BioLegend (1 mentions)
Cd326 g8, by BioLegend (1 mentions)
Anti ter119 ter 119, by BioLegend (1 mentions)
Dapi for nucleic acid staining, by Merck Group (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti cd135, by BioLegend (1 mentions)
5 protocols using anti annexin 5
1
Evaluating CD8+ T Cell Cytotoxicity
2
Apoptosis Profiling of iPSC-Derived Megakaryocytes
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Day 5 or 6 iPSC-derived megakaryocytes were stained for CD41a and CD42b, and then resuspended in 100 μL Annexin V Binding Buffer (BioLegend) with 5 μL anti–Annexin V and 5 μL 7-AAD (BioLegend). After incubation for 20 minutes at room temperature in the dark, cells were diluted with 400 μL Annexin V Binding Buffer and immediately analyzed by flow cytometry.
3
Analyzing Extracellular Vesicle Subtypes
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As previously described,19 10 μl S‐MVs were collected, and 1 μl of antibody was added. Antibodies included FITC labelled anti‐Annexin V (BioLegend,) and PE labelled EGFR (BioLegend,). In addition, Annexin V binding buffer (BioLegend,) was added as the protocol described. The percentages of subtypes of S‐MVs were gated according to the control group (BD LSRFortessa, Wuhan Institute of Virology). For the cellular uptake assay, the PKH67 labelled MVs treated CAL27 cells were collected and tested using FITC channel. The FITC positive group among total cells were gated using Flow Jo software.
4
Multimodal Isolation of Murine Organ Cells
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The lung, liver, spleen, kidney, and heart were extracted after mice were perfused with 1X PBS via the right atrium. For the heart, we used collagenase IV (10 mg/mL) as a digestive enzyme. For the spleen, no digestive enzymes were needed. The other tissues used collagenase type I, collagenase XI, and hyaluronidase. For the heart, we used collagenase IV. The cell suspensions were filtered through a 70-μm sterile nylon mesh cell strainer, washed with 1X PBS, and transferred to Eppendorf tubes. Fc receptors were blocked using TruStain fcX™ anti-mouse CD16/32 (BioLegend). The following antibodies were used: clone 6D5, 17A2, N418, M1/70, FA11, 30-F11, and 390. We also used anti-TER-119 (TER-119, BioLegend), anti-Annexin V (BioLegend), DAPI for nucleic acid staining (Sigma-Aldrich), PE anti-mCD47 (miap301, BioLegend), CD326 (G8.8, BioLegend), anti-Sca-1 (D7, BioLegend), anti-CD24 (M1/69, BD Biosciences), anti-CD271 (ME20.4, Invitrogen), and Monorab™ Rabbit Anti-Camelid VHH antibody, mAb (96A3F5, GenScript). Each stain was added at a 1:200 dilution to the cell suspension. Flow gating is shown (SI Appendix, Figs. S18 and S19 ). When Ai14 mice were used to quantify delivery, we also used PBS-treated Ai14 mice to control for flow gating.
5
Immunoblotting and Flow Cytometry Antibodies
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The following antibodies were used for immunoblotting: anti-phospho-STAT5 (#9351), anti-phospho-AKT (#9271), anti-phospho-ERK1/2 (#9370), anti-AKT (#4685), anti-ERK1/2 (#9102), anti-MCL1 (#5453), anti-histone H3 (#9717), and anti-acetylated histone H3 (#9677). All antibodies were all acquired from Cell Signaling Technology, Frankfurt, Germany. Anti-FLT3 (sc-480), anti-acetyl histone h4 (sc-377520) anti-beta actin (sc-8432), anti-Hsp90 (sc-101494), anti-STAT5 (sc-835), anti-GAPDH (sc-20357), and anti-goat IgG HRP secondary antibody (sc-2345) were purchased from Santa Cruz Biotechnology, Heidelberg, Germany while anti-rabbit and anti-mouse IgG HRP secondary antibody were purchased from Promega, Madison, WI, USA.
For flow cytometry, the anti-CD135 (#313305), anti-Annexin V (#640920), and human IgG1 PE isotype control (#403504) were purchased from BioLegend, San Diego, CA, USA.
For flow cytometry, the anti-CD135 (#313305), anti-Annexin V (#640920), and human IgG1 PE isotype control (#403504) were purchased from BioLegend, San Diego, CA, USA.
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