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Annexin 5 fitc 7 aad

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Annexin V-FITC/7-AAD is a combination of fluorescent labels used to detect and quantify apoptosis in cells. Annexin V binds to phosphatidylserine, which is exposed on the cell surface during early stages of apoptosis. 7-AAD is a dye that penetrates cells with compromised cell membranes, indicating late-stage apoptosis or necrosis. This combination provides a reliable method to differentiate between viable, early apoptotic, and late apoptotic/necrotic cells.

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7 protocols using annexin 5 fitc 7 aad

1

Apoptosis Pathway Regulation Assay

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Anti-MAT2A antibody (NBP1-92100, Rabbit polyclonal) was purchased from Novus Biologicals Company. Anti-PDCD6 antibody (12303-1-AP, Rabbit polyclonal) was purchased from Proteintech Company. Antibodies against Methylated-Lysine (ab23366, Rabbit polyclonal) and Pro-Caspase (ab32150, Rabbit monoclonal) were obtained from Abcam Company. Rabbit polyclonal antibody against Dimethyl-Arginine (07-414) was purchased from Millipore Sigma Company. Monoclonal antibody (F1804) that recognizes Flag was purchased from Sigma. Antibodies against β-actin (3700), Bcl-2 (15071), Bax (5023), Cleaved-Caspase 3 (9664), PARP (9532), Cleaved-PARP (5652), p-Acc (11818), p-JunB (8053) and p-MAPKAPK2 (3044) were purchased from Cell Signaling Technology. AMPK inhibitor compound C, SP600125 and SB203580 were obtained from Selleck Company. Cell counting kit 8 (CCK-8) was purchased from BestBio Science (Beijing, China). Annexin V-FITC/7-AAD was obtained from BD (Pharmingen, CA, USA). All other chemicals were obtained from commercially available. MG132 (protease inhibitor) and cycloheximide (CHX) were purchased from Sigma-Aldrich (USA).
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2

Apoptosis Quantification via Flow Cytometry

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5 × 105 cells/well in a 12-well plate were seeded. The next day, drugs were added to the well and incubated for 72 hours. The treated cells were trypsinized and resuspended in PBS. Cells were stained using the Annexin V-FITC/7-AAD (BD Pharmingen, USA) Kit as per the manufacturer's protocol. The stained cells were analysed on Beckman Coulter FC500 with a minimum of 10,000 events counted. Annexin V+/7-AAD- and Annexin V+/7-AAD+ cells were considered apoptotic cells.
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3

Flow Cytometry-based Apoptosis Assay

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Apoptosis was determined by flow cytometry using Annexin V-FITC/7AAD staining (BD PharMingen) after 72 h. The cellular potency of compounds as defined by half-maximal induction of apoptosis in primary CLL cells was determined using concentrations up to 100 μM. The fraction of viable cells was determined by counting annexin V/7-AAD double-negative cells for each individual dosage. Median values were subsequently applied for regression analysis and calculation of the half-maximal dosage effect (IC50). Curve fitting was performed using SigmaPlot (SPSS, Chicago). IC50 values were determined by fitting data to the Hill equation y = y0 + (axb)/(cb+xb).
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4

Measuring Cell Apoptosis and TUNEL Assay

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The cell apoptosis was measured using an Annexin V-FITC/7-AAD (#559763, BD Biosciences, NJ, USA) staining kit. HEI-OC1 cells were transfected with a REST plasmid or control vector, and then treated with H2O2. After treatment, cells were resuspended in binding buffer and stained with FITC-labeled Annexin V and 7-AAD, for 20 min at room temperature, in the dark. Cell apoptosis was detected using FACS Calibur (BD Biosciences, NJ, USA) and data were analyzed by using the FlowJo software v7.6.1 (FlowJo, LLC, NJ, USA).
Terminal Deoxynucleotidyl Transferase (TdT)-Mediated dUTP Nick-End Labeling (TUNEL) assay was used to detect the apoptotic cells, according to the manufacturer’s instructions. Cochlear sections were permeabilized in 0.3% Triton X-100 for 60 min, and then blocked with a fluorometric terminal deoxytransferase solution for 1 h, at 37 °C. The sections were then incubated with anti-Tuj1 primary antibody overnight at 4 °C. They were then stained with a Cy3-conjugated secondary antibody for 2 h, at room temperature. Finally, the nuclei were stained with DAPI, and fluorescent images were taken using a Leica TCS SP5 confocal fluorescent microscope.
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5

Evaluating NK Cell-Mediated Cytotoxicity

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NK cell lysis of target CRC cells was determined by ELISA and flow cytometry after 24 h of co-incubation with or without SHR-1316. Then to detect NK activation, the expression of soluble IFN-γ in culture was detected by enzyme-linked immunosorbent assay (ELISA). To study apoptosis, cells were stained with AnnexinV-FITC/7AAD (BD Biosciences, USA). The CD56-APC antibody (BD, Cat #555,518) was used to identify NK cells and the level of apoptosis was calculated for the CD56- cells. Next, to measure the level of NK cell degranulation, target cells and NK cells at 1:1 E/T ratio incubated with CD107a-PE (BD, Cat #555,801) for 4 h. The percentage of CD107a positive cells was calculated for the CD56+ cell fraction.
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6

Apoptosis Analysis of KYSE-30 and TE-1 Cells

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For apoptosis analysis, KYSE-30 and TE-1 cells were seeded in 6-well plates at a concentration of 1 × 106 cells/well with different agents for 48 h and divided into the four groups: control, anlotinib (20 μM), raltitrexed (2.5 μM) and anlotinib combined with raltitrexed (20 μM + 2.5 μM), the negative control for apoptosis analysis was the equivalent volume of DMSO. Apoptotic cells were distinguished by Annexin V-FITC/7AAD dual staining using apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA). Cells were collected and washed twice with cold PBS and centrifuged for 10 min at 20,000 rpm at 4 °C. Cells were cultured in 300 μL / 1 × 106 cells of 1 × annexin-binding buffer at room temperature for 5 min to prepare the cell samples for flow cytometry, and then double stained with annexin V-FITC (green) and 7AAD-PerCP (red) for 15 min. The percentage of apoptotic cells was analyzed with a FACS Calibur flow cytometer (BD Biosciences) using Flow Jo software (version 9.8.1). The apoptosis rate in every group was described by Q2 (late apoptosis) + Q3 (early apoptosis). All experiments were repeated for three times.
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7

Siglec-8-Mediated Eosinophil Apoptosis

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Purified eosinophils were suspended at 2×106/mL in culture medium in 96-well U bottom plates and incubated overnight at 37°C, 5% CO 2 with or without IL-5 (30 ng/mL) prior to a second overnight incubation with anti-Siglec-8 antibodies or their respective isotype controls. The addition of IL-5 to these cultures enhances the ability of Siglec-8 crosslinking by anti-Siglec-8 mAb as previously reported3 (link)–5 (link). The cells were subsequently washed, stained with Annexin-V-FITC/7-AAD (BD Biosciences) and acquired by flow cytometry (LSRII, BD).
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