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42 protocols using a00 1 1102

1

Analyzing HCOEPIC Apoptosis via Flow Cytometry

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To analyze HCOEPIC apoptosis, an Annexin V-FITC apoptosis detection kit (KGA108, KeyGEN, Nanjing, China) was used. According to the manufacturer's instructions, 500 μl of binding buffer was added to suspend the HCOEPICs, and then 5 μl of Annexin V-FITC was added. After mixing, 5 μl of propidium iodide was added and incubated with the cells at room temperature for 15 min. Cell apoptosis was analyzed via flow cytometry (A00-1-1102, Beckman, Brea, California, USA). The cells in the different groups were treated with 10 μM of DCFH-DA (mother liquid concentration: 10 mM) and incubated at 37°C for 20 min. The cells were washed three times with a serum-free medium and digested with trypsin. After cell collection, flow cytometry (A00-1-1102, Beckman) was used to detect reactive oxygen species (ROS) accumulation.
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ROS Levels Measurement Protocol

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The ROS level was measured using an ROS assay kit (S0033S; Beyotime). 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA; stock concentration, 10 mM) was diluted at 1 : 1000 in a serum-free medium to a final concentration of 10 μM. The treated cells (mentioned above) were removed from the cell culture medium, and DCFH-DA was added to cover the cells. Subsequently, the cells were again incubated at 37°C for 20 min. The samples were collected by trypsinization and flow cytometry (A00-1-1102; Beckman, USA) detection was performed.
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3

Annexin V Assay for Apoptosis

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Apoptosis was assessed using an Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, USA). After treatment, cells were harvested and washed twice with ice-cold PBS and binding buffer and incubated with AnnexinV-FITC in dark for at least 15 min. Propidium iodide (PI) was added in the dark for 10 min at room temperature. Apoptosis was determined by flow cytometry (A00-1-1102, Beckman, USA). The degree of early apoptosis was determined as the percentage of cells positive for Annexin V and negative for PI by flow cytometry. Cells staining negative for Annexin V and PI were considered to be normal. Cells staining positive for Annexin V and negative for PI represented early apoptosis. Finally, cells staining positive for both Annexin V and PI were classified as late apoptosis.
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Quantifying Intracellular Reactive Oxygen Species

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ROS detection kit (S0033S, Beyotime) was performed to measure ROS levels. 1 ​× ​106 ​cells were taken and centrifuged at 400 ​g for 5 ​min. DCFH-DA was diluted with the serum-free medium at 1:1000. The diluted DCFH-DA was added to suspend the cells. The cell precipitate was collected by centrifugation at 400g and washed with serum-free medium twice for flow cytometry (A00-1-1102, Beckman) detection.
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5

Quantification of Cellular Glucose Uptake

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According to the manufacturer instructions, the glucose uptake of HTR-8/SVneo cells was measured by a fluorescent glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-glucose (2-NBDG) (Invitrogen, N13195). Briefly, the cells were cultured in a medium with a 100 μM 2-NBDG for 1 hour at 37°C. The cells were washed twice with PBS, digested with trypsin, and centrifuged for 5 min at 1500 rpm at 4°C. Then, the cells were suspended in 200 μl of PBS in each tube and immediately analyzed and quantified by flow cytometry (A00-1-1102, Beckman, USA).
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6

Cell Cycle and Apoptosis Analysis

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To conduct cell cycle detection, the two cells were treated with 1.2 mL of pre-cooled 100% ethanol, resulting in a final concentration of 75%. Subsequently, the cells were fixed by overnight incubation at 4℃. After the cells were washed with PBS, they were centrifuged. Then, they were mixed with 150 µL of propidium iodide (PI, MB2920, Meilune, China) and stained for 30 min at 4℃. Different stages of the cell cycle have different DNA content, and PI can label DNA to determine which cycle the cell is in. Detection of stained cells with a flow cytometer (A00-1-1102, Beckman, USA), PI was excited by a 488 nm argon laser and detected through a 630 nm bandpass filter, collecting around 15,000 cells in FSC/SSC dot plots, and analyzing the percentage of cells in each cell cycle phase on the PI fluorescence histogram to determine positive cell staining. For apoptosis detection, SK-Hep-1 and HCC-LM3 cells were treated with trypsin (AWC0232, Abiowell, China). Following digestion, after washing the cells with PBS, approximately 3.2 × 105 cells were collected for further analysis. Following the instruction provided by the apoptosis detection kit (KGA1030, KeyGEN BioTECH, Nanjing, China), the cell suspension was treated with Annexin V-FITC (5 µL) and PI (5 µL) successively. The mixture was then incubated at room temperature for 10 min in the absence of light before further analysis.
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7

Quantification of α-SMA and S-100 Expression

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Samples were taken, each about 1 × 106 cells were placed in a 1.5 mL centrifuge tube, the cells were resuspended with 200 μL PBS volume, α-SMA (PA5-77846, eBioscience) and s-100 (MA5-32625, eBioscience) antibodies (5 μL for all three antibodies) were added respectively. The aforementioned cells were washed with 1 mL PBS, incubated in the dark for 30 min, and repeated twice. α-SMA and s-100 expressions were detected by flow cytometry (A00-1-1102, Beckman) after the cells were resuspended by adding 200 μL PBS, respectively.
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8

Lipid ROS and Mitochondrial Membrane Potential Analysis

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To detect lipid reactive oxygen species (ROS) levels in brain tissues and neuronal cells, the samples were processed in the following manner: Initially, they were digested with 0.25% trypsin to create cell suspensions. Following trypsin digestion, the cell suspensions underwent two washes with PBS. Post-washing, they were then immersed in 5 μM C11-BODIPY (MX5211-1 MG, MaokangBio, China) and incubated in a 37°C environment for 1 h. After this incubation period, cells were again washed and suspended in PBS, and the lipid ROS levels were subsequently measured via a flow cytometer (A00-1-1102, Beckman, USA).
For the assessment of mitochondrial membrane potential (MMP), additional steps were required. After the typical digestion and centrifugation processes, neuronal cells were treated with 0.5 mL of a specific JC-1 staining solution. They were then maintained in a 37°C cell culture incubator for a further 30 min. Subsequent to this incubation period, the neurons were washed and resuspended in a solution of JC-1 staining buffer, all set for MMP detection.
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9

Quantifying Lung Cell Immune Responses

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The expression level of Fcnb and the number of neutrophils and macrophages were detected by flow cytometry [40 ]. Lung tissue samples were digested to obtain cell suspension. Blood, BALF, and cell suspension were centrifugally stratified to obtain neutrophils and macrophages for re-suspension. Antibodies of LY-6G (11-9668-82, eBioscience, USA) and F4/80 (11-4801-82, eBioscience, USA) were added respectively to the suspensions of neutrophils and macrophages and incubated for 30 min away from light. Then, the mixture of Fcn B antibody (Ab70814, Abcam, UK) and Alexa Fluor 594-goat anti-rabbit IgG (A-11012, Invitrogen, USA) was added to the above cell suspensions and incubated for another 30 min away from light. After washing and suspension, the cells were detected by flow cytometry (A00-1-1102, Beckman, USA). In addition, ROS levels in lung tissues and MLE-12 were detected by flow cytometry. First, the tissue and cells were digested to obtain cell suspensions. The cell suspension was incubated with BODIPY™ 581/591 C11 fluorescent probe (A-11012, Invitrogen, USA) at 37 °C for 30 min and finally detected by flow cytometry.
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10

Apoptosis Detection by Flow Cytometry

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Apoptosis was detected according to the instructions of the apoptosis kit (KGA1030, KeyGEN BioTECH). Briefly, cells were digested with EDTA-free trypsin and washed with PBS to a concentration of 3.2 × 105 cells. Cells were suspended by adding 500 µL of Binding buffer. Subsequently, the percentage of apoptotic cells was assessed by flow cytometry (A00-1-1102, Beckman).
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