PBMCs were cultured with various concentrations of daunorubicin, tacrolimus, MPA and prednisolone (Sigma) and apoptosis detected by intracellular staining for activated caspase 3 (BD Pharmingen, Wokingham, UK) or by annexin V (Miltenyi Biotec) surface staining after 24 or 48 h, respectively. annexin V staining was performed for 15 min at room temperature in annexin V binding buffer (Miltenyi Biotec); 50 M Verapamil (Sigma) was added at the beginning of culture. Percentages expressing annexin V at each concentration were background subtracted for annexin V expression in media alone.
Annexin 5
Manufactured by Miltenyi Biotec
Sourced in Germany
Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine (PS). Annexin V can be used to detect apoptosis by binding to PS exposed on the cell surface during the process of programmed cell death.
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Lab products found in correlation
Propidium iodide, by Miltenyi Biotec (3 mentions)
Facscalibur, by BD (2 mentions)
Tacrolimus, by Merck Group (1 mentions)
Verapamil, by Merck Group (1 mentions)
Prednisolone, by Merck Group (1 mentions)
Activated caspase 3, by BD (1 mentions)
Annexin 5 binding buffer, by Miltenyi Biotec (1 mentions)
Cd11b, by Thermo Fisher Scientific (1 mentions)
B220 ra3 6b2, by Thermo Fisher Scientific (1 mentions)
Rb6 8c5, by Thermo Fisher Scientific (1 mentions)
Mhcii m5 114.15.2, by Thermo Fisher Scientific (1 mentions)
Ly6g 1a8, by BD (1 mentions)
Mr5d3, by Bio-Rad (1 mentions)
Cd206, by Bio-Rad (1 mentions)
C92 605, by BD (1 mentions)
Tcrb h57 597, by Thermo Fisher Scientific (1 mentions)
Lyve 1, by Abcam (1 mentions)
Siglec f, by BD (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
17 protocols using annexin 5
1
Apoptosis Assay of PBMCs
2
Quantifying Peritoneal Macrophage Subsets
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Two methods were used to quantify peritoneal macrophages. Total peritoneal cells were counted in a hemacytometer or using an automated cell counter. Then this number was multiplied by the percent of macrophages stained for CD115 (AFS98; eBioscience), ICAM-2 (3C4; BioLegend) and F4/80 (BM8, eBioscience) by flow cytometry. These antibodies allow us to distinguish the minor and major peritoneal macrophage populations (Gautier et al., 2012 (link)), with only the major macrophage population expressing ICAM-2 (Gautier et al., 2012 (link)). Alternatively, peritoneal cells obtained by lavage were incubated with FITC-conjugated anti–ICAM-2 mAb, and then a manual count of the fraction of ICAM-2+ cells was made and multiplied by the total number of peritoneal cells. Both methods yielded similar results. Other reagents and mAbs used in flow cytometry were Annexin V (Miltenyi Biotec) and antibodies against Ki67 (B56; BD), active caspase 3 (C92-605; BD), pH3 (D2C8; Cell Signaling technology), TIMD4 (RMT4-54; eBioscience), CD206 (MR5D3; Serotec), LYVE-1 (Abcam), Siglec F (E50-2440; BD), CD11b (M1/70; eBioscience), CD5 (53–7.3; BD), B220 (RA3-6B2; eBioscience), ly 6C/G (RB6-8C5; eBioscience), Ly6-G (1A8; BD), TCRb (H57-597; eBioscience), MHC-II (M5/114.15.2; eBioscience), and MARCO (ED31; Serotec).
3
Apoptosis-Mediated TREM2 Activation in Neurodegeneration
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Phosphatidylserine (PS) expression on the external surface of apoptotic SH-SY5Y cells was used as a ligand for TREM2 as we previously reported10 (link). Briefly, SH-SY5Y cells (a kind gift from Prof R de Silva, University College London, Queen Square Institute of Neurology) were cultured in DMEM with 10% FBS (Life Technologies) and 1% penicillin/streptomycin (Life Technologies). To induce apoptosis, SH-SY5Y cells were heat shocked at 45 °C for 2 h, before resuspension in iPS-Mg medium. Cell death and exposure of cell membrane PS was confirmed by flow cytometry using FITC-conjugated Annexin-V (Miltenyi Biotec) and propidium iodide staining. Apoptotic cells (“PS+ cells”) were added to iPS-Mg at a ratio of 2:1 PS+ cells:iPS-Mg for either 5 min (signalling assays) or O/N (inflammasome assays). Exposure of iPS-Mg to PS+ cells was blocked by preincubation of PS+ cells for 1 h with 5 µg recombinant Annexin V (268–10,001, RayBiotech). SYK signalling was blocked by preincubation of iPS-Mg for 1 h with 10 µM BAY61-3605 prior to addition of PS+ cells.
4
Apoptosis Quantification in RB Cells
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RB cells (1 × 106 cells) were treated with inhibitors for 48 h and stained with Annexin V and Propidium Iodide (PI) as per manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Stained cells were immediately analyzed on a cytoFLEX flow cytometer (Beckman Coulter Life Sciences, Brea, CA, USA).
5
Intestinal Organoid Proliferation and Apoptosis after Viral Infection
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Proliferation in intestinal organoids was measured by EdU (5-ethynyl-2′-deoxyuridine) incorporation after H9N2 virus infection. In brief, EdU (1:2000) was pre-incubated in ENR-medium for 2 h. The nuclei were stained by Hoechst33342. Cells were then detected with a fluorescence microscope.
Apoptotic cells were detected by Annexin V and propidium iodide (PI) staining assay (Miltenyi Biotec, Shanghai, China) according to the manufacturer’s protocols. Briefly, organoids were digested with accutase (Millipore) for 25 min at 37 °C and single-cell suspensions were obtained. Cells were harvested and washed with PBS. Following this, the cells were incubated with 5 μL Annexin V-FITC and 5 μL PI-FL3 for 10 min and detected with FACS Calibur (Becton, Dickinson and Company, USA). Single cell was gated using FSC and SSC parameters and apoptotic cells of organoids were analyzed at FL-1 and FL-3 by FACS.
Apoptotic cells were detected by Annexin V and propidium iodide (PI) staining assay (Miltenyi Biotec, Shanghai, China) according to the manufacturer’s protocols. Briefly, organoids were digested with accutase (Millipore) for 25 min at 37 °C and single-cell suspensions were obtained. Cells were harvested and washed with PBS. Following this, the cells were incubated with 5 μL Annexin V-FITC and 5 μL PI-FL3 for 10 min and detected with FACS Calibur (Becton, Dickinson and Company, USA). Single cell was gated using FSC and SSC parameters and apoptotic cells of organoids were analyzed at FL-1 and FL-3 by FACS.
6
Apoptosis Assessment of Human EPCs
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Human EPCs (CD34+) following hyperglycemia (20 mmol/L glucose) exposure were stained with propidium iodide (Miltenyi Biotec, 130‐093‐233) and annexin V (Miltenyi Biotec, 130‐097‐928) along with suggested isotype control using Miltenyi Biotec standard protocols. Both human EPCs (CD34+) and HUVECs were stained with propidium iodide. The cells were analyzed poststaining using BD FACSCalibur (BD Biosciences) cell analyzer.
7
Magnetic Separation and Fluorescent Labeling
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For magnetic separations, annexin V, CD45 and CD235a magnetic beads; and for fluorescent labeling, CD45-FITC fluorophore were purchased from Miltenyi Biotec. For buffer preparation, chemicals were purchased from Sigma Aldrich (Sigma Aldrich, St. Louis, MO, USA). TUNEL assay for DNA-fragmentation determination was from Thermo Fisher Scientific (Thermo Fisher Scientific, Waltham, MA, USA). For density gradient centrifugations, PureCeptionTM and sperm preparation medium were purchased from Origio, CooperSurgical (CooperSurgical, Trumbull, CT, USA).
8
Annexin V Apoptosis Assay
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Cells were seeded in a 60% confluence and 12 h later treated with 5 µM purvalanol A for 24 h. Cells were harvested, washed twice with PBS-3 mM EDTA, resuspended in 100 µL of Binding Buffer (10 mM Hepes/NaOH, 140 mM NaCl and 2.5 mM CaCl2, pH 7.4) and 2 µL FITC Annexin V (BD Bioscience) and incubated for 30 min at 4 °C. Cells were washed twice with PBS and resuspended in 250 µL of PBS. Annexin V binding was assayed in MACSQuant VYB (Miltenyi Biotec) and the results were analyzed with Flow Logic software (Miltenyi Biotec).
9
Monocyte Extracellular Surface Staining
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Antibodies used for extracellular staining of monocytes and their surface molecules were purchased from BD Pharmingen, Heidelberg, Germany (CD14-PE (clone M5E2) and Annexin V) and from Miltenyi Biotec, Bergisch Gladbach, Germany (CD11b-PE (clone REA731), CD14-APC (clone TÜK), CD18 (clone TS1/18)). All antibodies were tested for their specificity by isotype control staining, when introduced to our laboratory. Data acquisition was performed with a FACSCanto II flow cytometer (BD Bioscience) and data were analyzed via FlowJo V10 (FlowJo, LLC, Ashland, OR). To assess the expression level of GFP, Rhodamine, CD11b and CD18 on monocytes, we used the geometric mean fluorescence intensity (gMFI).
10
Multiparametric analysis of T-cell activation
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Antibodies against the following proteins were purchased from BD Biosciences: CD3 (HIT3a and UCHT1), CD69 (SK7), CD25 (M-A251), CD28 (CD28.2), pSTAT5 pY694 (clone 47), p38MAPK pT180/pY182 (38/p38), TNFα (Mab11), IFN-γ (B27), and Annexin V. Antibodies against the following proteins were purchased from Miltenyi Biotech: CD8 (BW135/80), CD45RA (T6D11), CCR7 (REA108), pAKT pS473 (REA359), and pERK1/2 pT202/pY204 (REA152). Antibodies against the following proteins were purchased from Cell Signaling Technology: pBAD S112 (40A9), p70 S6K pT389 (D57.2.2E), and p4EBP1 pT37/46 (236B4). IL-15 was purchased from Miltenyi Biotech. 2-Deoxy-d -glucose (2-DG), 6-Diazo-5-oxo-l -norleucine (DON), oligomycin, PD98059, SB203580, and LY294002 were obtained from Sigma. Granzyme B inhibitor IV was obtained from Calbiochem.
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