Peritoneal cells were harvested from BALB/c mice. Analyses of apoptosis were performed as described elsewhere (34 (link)). The cells were then plated in 24-well plates at a density of 1 × 106 cells per well. If necessary, SNA (10 μg/ml) pretreatment was applied. A/WSN/1933 virus or UV-WSN virus was added to each well at a 1 × 106 pfu. After incubation at 37°C for different time periods, cells were harvested and stained with anti-FcγRII/III (Fc receptor blocker) before being stained with APCef780-conjugated anti-CD19 (Catalog No: 47-0193-80, eBioscience), PE-conjugated anti-CD23 (Catalog No: 12-0232-81, eBioscience), FITC-conjugated anti-B220 (Catalog No: 553088, BD Biosciences), FITC-conjugated anti-F4/80 (eBioscience), and BV421-conjugated anti-CD11b antibodies (Catalog No: 48-0112-80; eBioscience). After 1 h incubation at 4 °C, cells were washed with FACS buffer and stained with APC-conjugated Annexin V (Catalog No: 17-8007-74, eBioscience) for 15 min at room temperature. To exclude dead cells, stained cells were incubated with 10 μg/mL of PI (eBioscience, San Diego, CA, USA) or 7AAD (Catalog No: 51-68981E, BD Biosciences). The cells were analyzed with a FACSCantoTM II.
Apc conjugated annexin 5
Manufactured by Thermo Fisher Scientific
Sourced in United States
APC-conjugated Annexin V is a fluorescent labeling reagent used for the detection of apoptotic cells. Annexin V is a calcium-dependent phospholipid-binding protein with a high affinity for phosphatidylserine, which is exposed on the surface of apoptotic cells. The APC (Allophycocyanin) fluorescent label allows for the visualization and quantification of apoptotic cells by flow cytometry or other fluorescence-based techniques.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Lsrfortessa flow cytometer, by BD (2 mentions)
Trypsin edta, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by BD (2 mentions)
Annexin 5 binding buffer abb, by Thermo Fisher Scientific (2 mentions)
Cyan adp flow cytometer, by Agilent Technologies (2 mentions)
7 aad, by BD (1 mentions)
Fitc conjugated anti b220, by BD (1 mentions)
Fitc conjugated anti f4 80, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti cd23, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Caspase glo 3 7 assay, by Promega (1 mentions)
Csampler software, by BD (1 mentions)
Celltiter glo, by Promega (1 mentions)
Canto 2, by BD (1 mentions)
Cd11c apc hl3, by BD (1 mentions)
Cytofix cytoperm buffer, by BD (1 mentions)
Cd8 percpcy5.5 53 6 7, by Thermo Fisher Scientific (1 mentions)
Ly 6g pe cy7 1a8, by BioLegend (1 mentions)
Q vd oph, by R&D Systems (1 mentions)
18 protocols using apc conjugated annexin 5
1
Apoptosis Analysis of Peritoneal Cells
2
In Vivo Cytolabeling of Cells in ACE Grafts
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In some instances, fluorescently conjugated antibodies were injected directly into ACE for in vivo cytolabeling in situ. With our intravital imaging platform, the injected antibodies can effectively label cells up to 50 µm deep within the graft tissue, which is at the similar depth capacity for accurately tracking cells marked with genetic tags of fluorescence markers (Abdulreda et al., 2011 (link)). After mice were adoptively transferred with Treg and Teff cells, mice were injected with fluorochrome-conjugated antibodies directly into the anterior chamber. All antibodies were tested with isotype controls to determine specificity of the in situ labeling. The following is a list of the antibodies and their isotype controls. BV605 conjugated anti-CD11b monoclonal antibody or rat Ig2a isotype control; Alexa Fluor 647–conjugated anti-CD11c, anti-CD80, and anti-CD86 monoclonal antibodies and hamster IgG isotype control (BioLegend). APC-conjugated Annexin V (eBioscience) was used to assess apoptotic cells in ACE grafts. This in vivo assay for β cell apoptosis was described previously (Speier et al., 2008a (link),b (link)).
3
Cell Cycle and Cell Death Analysis
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For cell cycle analysis, cells were seeded in 6-wells plates and cultured until 70–80% confluence. Cells were incubated with ACF (in supplemented serum-free RPMI 1640 medium) for 24 or 48 hours, after which cell cycle analysis was performed using flow cytometry according to ref. 69 .
The mode of cell death following ACF incubation was analyzed by flow cytometry using APC-conjugated Annexin V (eBioscience, San Diego, CA) for apoptosis and TO-PRO-3 (Life Technologies) for necrosis. Cells were seeded in 6-wells plates and cultured until 70–80% confluence. Next, cells were incubated with ACF (in supplemented serum-free RPMI 1640 medium) for 24 or 48 hours. After incubation, the samples were prepared as described previously [36 (link)] and assayed on a FACSCanto II (Becton Dickinson, Franklin Lakes, NJ). Ten thousand events were recorded in the gated region and data was analyzed using FlowJo software (Treestar, Ashland, OR).
The mode of cell death following ACF incubation was analyzed by flow cytometry using APC-conjugated Annexin V (eBioscience, San Diego, CA) for apoptosis and TO-PRO-3 (Life Technologies) for necrosis. Cells were seeded in 6-wells plates and cultured until 70–80% confluence. Next, cells were incubated with ACF (in supplemented serum-free RPMI 1640 medium) for 24 or 48 hours. After incubation, the samples were prepared as described previously [36 (link)] and assayed on a FACSCanto II (Becton Dickinson, Franklin Lakes, NJ). Ten thousand events were recorded in the gated region and data was analyzed using FlowJo software (Treestar, Ashland, OR).
4
Cell Viability, Cytotoxicity, and Apoptosis Assays
5
Multiparameter Flow Cytometry Analysis
6
Annexin V Apoptosis Assay
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Cells were washed in PBS then harvested by trypsinization and resuspended in an Annexin binding buffer containing APC conjugated Annexin V (Ebioscience, San Diego, CA, USA) plus propidium iodide. Cells were then analyzed by flow cytometry, and percentages of positive cells were calculated using Flowjo software.
7
Diaryl thiazoline and thiazole compounds
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Fluorizoline (a diaryl trifluorothiazoline; see molecular structure in Figure 1A ) and compound 2a (a non-fluorinated diaryl thiazole synthetic precursor of fluorizoline; see molecular structure in Supplementary Figure 2A ) were synthesized as previously described [12 ] and dissolved in DMSO at 20 or 100 mM. Allophycocyanin (APC)-Cy7-labeled antibody against human CD11b was purchased from BD Biosciences (Franklin Lakes, NJ, USA). APC-Alexa Fluor 750 anti-human CD33 was purchased from Beckman Coulter (Marseille, France). APC-Cy7 anti-human CD34 was from Sony Biotechnology Inc. (Champaing, IL, USA). APC-conjugated annexin V was purchased from eBiosciences (San Diego, CA, USA). Q-VD-OPh was from R&D Systems (Minneapolis, MN, USA). MTT ((3-4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) was from Sigma-Aldrich. PI was purchased from eBiosciences (San Diego, CA, USA).
8
Analyzing Apoptosis via Annexin V and PI
9
Apoptosis Quantification by Flow Cytometry
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Apoptosis was assessed by AnnexinV/PI staining. Cells (2 × 105) were seeded in a 6-well plate and treated with or without 50 ng/ml dox the next day. After 5 days, cells and their supernatant were collected and washed with cold PBS. Cells were stained for AnnexinV and PI by incubation in 100 μl staining solution (1 μl APC-conjugated AnnexinV (eBioscience), 0.1 μg PI (Invitrogen), 1× binding buffer) for 15 min at room temperature in the dark. Next, cells were resuspended in 1× binding buffer and results were immediately acquired on a FACSCaliburTM flow cytometer (BD Biosciences) and analyzed using FlowJo® software (version 10.4.1). Three independent experiments were performed.
10
Annexin-V Staining for iPSC and iNCC Apoptosis
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Cell apoptosis was assessed by annexin-V staining. For assessment of iPSCs, iPSC colonies were grown to approximately 60% confluency on VTN-treated 6-well tissue culture plates. For iNCCs, 120h differentiated iNCCs cultured on VTN-treated 6-well tissue culture plates were used. Cells were disrupted using trypsin-EDTA (Sigma) treatment and cells pelleted by centrifugation. Cells were washed once in PBS and then washed once in 1X Annexin-V Binding Buffer (ABB) (Invitrogen). The cells were resuspended at a density of 1 x 106 cells/ml in 1X ABB, and 7.5μl APC-conjugated Annexin-V (Invitrogen) added to 100μl cell suspension. The cells were incubated for 20min at room temperature in the dark, washed in 1X ABB and resuspended in 200μl 1X ABB. 0.25μg/ml DAPI (Invitrogen) was added and the resulting samples immediately analysed by flow cytometry. Subpopulations were ascertained in a BD LSRFortessa flow cytometer (gated using an unstained control) as follows: live cells (annexin V-, DAPI-); necrotic cells (annexin-V-, DAPI+); early apoptotic (annexin V+, DAPI-); late apoptotic (annexin V+, DAPI+). Early and late apoptotic subpopulations were pooled for analysis.
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