Apoptosis was measured using the FITC-Annexin V Apoptosis Detection kit (BD Biosciences) as previously described (24 (link)). BMDMs were harvested by centrifugation at 1,000 x g for 5 min at 4˚C and washed twice with ice-cold PBS. The cells were resuspended in binding buffer [10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2] at a concentration of 1x106 cells/ml, and then gently mixed and incubated with 5 µl Annexin V-FITC (BD Biosciences) and 10 µl propidium iodide (PI; BD Biosciences) in the dark at room temperature for 15 min. After washing the cells with 1X binding buffer to remove the excess FITC-Annexin V and PI, apoptotic cells were analyzed using flow cytometry (FACSVia Flow Cytometer; BD Biosciences) within 1 h to determine the levels of apoptosis. The fluorescence of FITC and PI were measured using the FL-1 and FL-2 channels, respectively. Apoptosis was quantified by the percentage of the population shifting to fluorescence positivity. The percentage of apoptotic cells was calculated as the percentage of early and late apoptotic cells. The data were analyzed using CytExpert software 2.0 (Beckman Coulter, Inc.).
Cytexpert software 2
Manufactured by Beckman Coulter
Sourced in United States
CytExpert software 2.0 is a comprehensive software package designed for data acquisition, analysis, and management of flow cytometry experiments. It provides a user-friendly interface for researchers and laboratory professionals to efficiently process and interpret flow cytometry data.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Merck Group (3 mentions)
Flow cytometry, by Beckman Coulter (3 mentions)
Cytoflex, by Beckman Coulter (3 mentions)
Cytoflex flow cytometer, by Beckman Coulter (3 mentions)
Mouse brain slicer matrix, by Zivic Instruments (2 mentions)
Annexin 5 fitc apoptosis detection kit, by Beyotime (2 mentions)
Annexin 5 fitc, by BD (1 mentions)
Facsvia flow cytometer, by BD (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Cytoflex cytometer, by Beckman Coulter (1 mentions)
Cd11b fitc, by Miltenyi Biotec (1 mentions)
Ly6g apc, by BD (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Trizol reagent, by Qiagen (1 mentions)
Acsa 2 apc, by Miltenyi Biotec (1 mentions)
Evos fl auto cell imaging system, by Thermo Fisher Scientific (1 mentions)
Myelin removal kit, by Miltenyi Biotec (1 mentions)
Sytox aadvanced dead cell stain, by Thermo Fisher Scientific (1 mentions)
Cytoflex s cytometer, by Beckman Coulter (1 mentions)
Rnase a, by Beyotime (1 mentions)
23 protocols using cytexpert software 2
1
Quantifying Apoptosis using Annexin V-FITC
2
Resveratrol Effect on HUVEC Apoptosis
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H2O2-induced HUVEC cells were seeded in 6-well plates (1 × 105 cells/well) and were then treated with resveratrol or R-PC. Next, cells were stained with 2 μL Annexin V-FITC and 10 μL PI. Samples were measured using flow cytometry (Beckman Coulter, Brea, CA, USA). Finally, the cell apoptosis number was analyzed by CytExpert software 2.0 (Beckman Coulter, Inc., Brea, CA, USA).
3
Isolation and Analysis of Brain Immune Cells
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On day 1 after ICH, mice were anesthetized with 3% isoflurane and were perfused with cold phosphate-buffered saline. Brains were cut into 1-mm-thick coronal sections with a mouse brain slicer matrix (Zivic Instruments, Pittsburgh, PA). Four 1-mm-thick brain sections of the left striatum with hemotoma were collected. The striatum was dissociated with GentleMACS Dissociator (Miltenyi Biotec, Auburn, CA) as described previously28 (link),30 (link). The final cell suspension were incubated with the primary antibodies: CD11b-FITC (Miltenyi Biotec, 130–113-234) and ACSA-2-APC (Miltenyi Biotec, 130–117-535) or and Ly6G-APC (Pharmingen, 560,599) for 30 min at 4ºC. The corresponding isotype antibodies were used as negative control. Propidium iodide (Sigma, St. Louis, MO) staining was used to exclude dead cells. Cell supernatants were analyzed by CytoFLEX cytometer (Beckman Coulter, Indianapolis, IN) with CytExpert software 2.0 (Beckman Coulter). Macrophage/microglia cells were distinguished as CD11b-FITC+ / ACSA-2-APC- population. Astrocytes were distinguished as CD11b-FITC- / ACSA-2-APC+ population. Neutrophils were distinguished as CD11b-FITC+ / Ly6G-APC+ population. Gated cells were collected into TRIzol reagent (Qiagen, 217,004) for mRNA extraction and real-time PCR.
4
Analysis of Neural Crest Stem Cells
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NCSCs from control hiPSCs and SOX10-KO hiPSCs were isolated or examined by FACS. Cells were dissociated with Accutase into single-cell suspensions and incubated with monoclonal antibodies against human antigens, including p75, HNK1, or CD49D (all from BD Biosciences). An irrelevant isotype-identical antibody (BD Biosciences) served as a negative control. Data were analyzed using CytExpert software 2.0 (Beckman Coulter, CA, USA). Information of the antibodies is listed in Supplementary Table 4 .
5
Annexin V-FITC Apoptosis Assay in AGS Cells
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The effects of BQ and OQ on the apoptosis of AGS cells were quantitated using the Annexin V-FITC Apoptosis Detection Kit (Beyotime Institute of Biotechnology) and flow cytometry. AGS cells were treated with BQ, OQ, or 5-FU at a concentration of 3 µM for 3, 6, 12 or 24 h. A volume of 200 µl binding buffer was incubated with AGS cells, followed by staining with 3 µl Annexin V-FITC and 2 µl propidium iodide (PI) at the 4°C for 30 min. The stained AGS cells were observed using the EVOS FL Auto Cell Imaging System (Thermo Fisher Scientific, Inc.) at a magnification of ×400, and analyzed using a flow cytometer (Beckman Coulter, Inc.). Data were analyzed using CytExpert software 2.0 (Beckman Coulter, Inc.).
6
Intracellular ROS Quantification in AGS Cells
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To determine the intracellular levels of ROS, DCFH-DA was utilized. AGS cells were plated in 6-well plates and grown to 60% confluence. AGS cells were treated with 3 µM BQ or OQ for 3, 6, 12 or 24 h. Cells were harvested, stained with 10 µM DCFH-DA for 30 min at 37°C, washed twice with PBS, and then immediately analyzed by flow cytometry (Beckman Coulter, Inc.). ROS levels were analyzed using CytExpert software 2.0 (Beckman Coulter, Inc.).
7
Isolation of Microglia from Mouse Striatum
8
Androgen-Dependent TF Expression
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LNCaP cells were stimulated with different concentrations of DHT (1–100 nm ) for 48 h, harvested by scraping, and finally fixed in 1% paraformaldehyde. To determine the total cellular TF protein content in LNCaPs, cells were permeabilized with 0.1% Triton X‐100 in phosphate‐buffered saline (PBS) for 15 min, and labeled with anti‐CD142–fluorescein isothiocyanate (anti‐TF) (CLB/TF5; Cat. No. MA1‐82810; Thermo Fisher Scientific). Mean fluorescence intensity (MFI) was analyzed with a BD Accurri C6 flow cytometer and BD Accuri C6 Samples software (Becton Dickinson, Schwechart, Austria). MyC‐CaP cells were stimulated with different concentrations of DHT (1–100 nm ) for 48 h, and detached with Versene solution at 4 °C (0.5 mm EDTA in PBS). To determine the surface TF protein content, MyC‐CaP cells were labeled with anti‐TF–phycoerythrin (R&D Systems, Minneapolis, MN, USA: Fab3178P). Live cells were separated by staining with SYTOX AADvanced Dead Cell stain (Thermo Fisher Scientific). MFI was analyzed with a Cytoflex S cytometer and Cytexpert software 2.0 (Beckman Coulter, Vienna, Austria).
9
Cell Cycle Analysis of MCF-7 Cells
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MCF-7 cells were treated with 10 µmol/l quinalizarin for 3, 6, 12 and 24 h at a density of 1×105 cells/per well. Subsequently, the cell culture RPMI-1640 medium (10% FBS; 100 U/ml penicillin; 100 µg/ml streptomycin) was removed, and the cells were trypsinized (0.05% trypsin-EDTA in PBS), washed twice with cold PBS and fixed in 70% ethanol for 12 h at −20°C. Cell suspensions were then incubated with RNase A and PI (both Beyotime Institute of Biotechnology) for 30 min at 37°C in the dark. The stained cells were analyzed for DNA content using flow cytometry (Beckman Coulter, Inc.). The cell cycle was analyzed using CytExpert software 2.0 (Beckman Coulter, Inc.).
10
Apoptosis Analysis of MCF-7 Cells
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Early and late apoptosis were analyzed by flow cytometry. MCF-7 cells were seeded onto cell slides in 6-well plates (1×105 cells/well) and treated with 10 µmol/l 5-FU or 10 µmol/l quinalizarin at 3, 6, 12 and 24 h, as aforementioned. Cells were centrifuged at 5,000 × g for 5 min at 4°C and washed three times with PBS. A total of 10 µl FITC and 5 µl PI were incubated with the cell suspension for 20 min at 37°C in the dark and analyzed by flow cytometry (Beckman Coulter, Inc. Brea, CA, USA). CytExpert software 2.0 (Beckman Coulter, Inc.) was used for analysis.
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