The H9c2 cell culture medium was sucked out into a suitable centrifuge tube. Then the adherent H9c2 cells were washed by PBS once and digested by trypsin cell digestion solution, which contained EDTA (Solarbio, T1300-100, Beijing, China). After incubation at room temperature, we added the cell culture medium which was collected before, and transfered it to the centrifugal tube, then centrifuged 1000 g for 5 min (Bioridge, TDZ4-WS, Shanghai, China). The H9c2 cells were centrifuged and resuspended with 195 μl Annexin V-FITC binding solution and 5 μl Annexin V-FITC (Beyotime, C1062, Nantong, China), incubated at 4°C for 15 min. Then, the cells were centrifuged again and resuspended with 190 μl Annexin V-FITC binding solution and 10 μl propidium iodide staining solution (Beyotime, Nantong, China), incubated at 4°C for 5 min. The fluorescence was detected by flow cytometer (BD, Accuri C6, United States).
C1062
Manufactured by Beyotime
Sourced in China
The C1062 is a laboratory equipment product. It is designed for general laboratory use, but its core function is not specified in the information provided. A more detailed and unbiased description cannot be given without the risk of extrapolation or interpretation.
Automatically generated - may contain errors
Lab products found in correlation
Beckman cytoflex s, by Beckman Coulter (3 mentions)
Propidium iodide staining solution, by Beyotime (1 mentions)
T1300 100, by Solarbio (1 mentions)
Annexin 5 fitc binding solution, by Beyotime (1 mentions)
Accuri c6, by BD (1 mentions)
Cytoflex 1, by Beckman Coulter (1 mentions)
Propidium iodide, by BD (1 mentions)
Goat anti rabbit secondary antibody, by Beyotime (1 mentions)
Quickblock blocking buffer, by Beyotime (1 mentions)
9 protocols using c1062
1
H9c2 Cell Apoptosis Assay
2
Apoptosis and Necrosis Quantification
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An Annexin V-FITC and propidium iodide (PI) double staining assay was used to measure apoptotic and necrotic cells by flow cytometry according to the manufacturer's instructions (C1062, Beyotime). Briefly, 1 × 106 cells were collected by trypsinization, washed twice with ice-cold PBS, and resuspended in 200 μl binding buffer containing 10 μl Annexin V and 5 μl PI. After incubation for 30 min on ice in the dark, cells were analyzed using a FACSCalibur flow cytometer.
3
Evaluating ZIF-8 Rescue of TPEN-Induced Apoptosis
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The flow cytometric analysis was used to determine The recovery ability of ZIF-8 to TPEN-induced apoptosis. Briefly, the DPSCs were pretreated with a concentration gradient of ZIF-8 overnight, and then TPEN (2 µM) was added into the medium. After 24 h, the control and treated cells were collected and counted. According to the specification of the detection kit (Beyotime, C1062), 106 cells were taken and stained with annexin V and PI. The apoptosis was analyzed applying a Beckman CytoFLEX S flow cytometer (Beckman Coulter, CA, USA).
4
Evaluating ZIF-8 Rescue of TPEN-Induced Apoptosis
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The flow cytometric analysis was used to determine The recovery ability of ZIF-8 to TPEN-induced apoptosis. Briefly, the DPSCs were pretreated with a concentration gradient of ZIF-8 overnight, and then TPEN (2 µM) was added into the medium. After 24 h, the control and treated cells were collected and counted. According to the specification of the detection kit (Beyotime, C1062), 106 cells were taken and stained with annexin V and PI. The apoptosis was analyzed applying a Beckman CytoFLEX S flow cytometer (Beckman Coulter, CA, USA).
5
Cell Cycle and Apoptosis Analysis
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For EdU staining, 1 × 105 cells were seeded into 6-well plates. After the cell density reached about 90%, EdU (10 μM, C0088S, Beyotime) was added, and the staining procedure was operated following the manufacturer’s instructions. The cell cycle status were analyzed by flow cytometry (CytoFLEX 1, Beckman Coulter, Brea, CA, USA). For cell apoptosis analysis, 1 × 105 cells were seeded into 6-well plates. Before the cell density reached about 90%, the cells were collected and stained with Annexin V and propidium iodide (PI) (C1062, Beyotime), and then analyzed by flow cytometry (CytoFLEX 1).
6
Annexin V-FITC and PI Apoptosis Assay
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For the apoptosis assay, the proportion of apoptotic cells was evaluated by dual staining with Annexin V-FITC and PI (Beyotime C1062, Shanghai, China). A combination of Annexin V-FITC and PI staining distinguished early apoptotic cells (Annexin V+, PI−) and late apoptotic cells (Annexin V+, PI+). The apoptosis of all stained cells was analyzed using a flow cytometer (BD Biosciences, San Jose, CA, USA).
7
Evaluating ZIF-8 Rescue of TPEN-Induced Apoptosis
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The flow cytometric analysis was used to determine The recovery ability of ZIF-8 to TPEN-induced apoptosis. Briefly, the DPSCs were pretreated with a concentration gradient of ZIF-8 overnight, and then TPEN (2 µM) was added into the medium. After 24 h, the control and treated cells were collected and counted. According to the specification of the detection kit (Beyotime, C1062), 106 cells were taken and stained with annexin V and PI. The apoptosis was analyzed applying a Beckman CytoFLEX S flow cytometer (Beckman Coulter, CA, USA).
8
Cell Cycle and Apoptosis Analysis
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For cell cycle analysis, cells transfected with the indicated siRNAs were collected by trypsinizing gently and fixed in 70% ethanol overnight at −20°C. Cells were stained with Propidium Iodide (550825; BD Biosciences) for 15 min and analyzed by flow cytometry.
For cell apoptosis analysis, cells transfected with the indicated siRNAs were treated with AZD7762, PF477736, and LY2603618 respectively for 24 h, followed by staining with Annexin V-FITC/Propidium Iodide (PI) using an apoptosis detection kit (C1062; Beyotime) according to the manufacturer’s instructions. Subsequently, the cells were analyzed by flow cytometry.
For cell apoptosis analysis, cells transfected with the indicated siRNAs were treated with AZD7762, PF477736, and LY2603618 respectively for 24 h, followed by staining with Annexin V-FITC/Propidium Iodide (PI) using an apoptosis detection kit (C1062; Beyotime) according to the manufacturer’s instructions. Subsequently, the cells were analyzed by flow cytometry.
9
Immunofluorescence and TUNEL Assay for Pulmonary Apoptosis
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Slices (thickness: 5-μm) mentioned above were used for immuno uorescence staining. After boiling with sodium citrate at 100℃ for 20 min, the cooled sections were incubated with 1% Triton X-100 for 20 min and blocked with the QuickBlock TM Blocking Buffer (P0260, Beyotime) at 25℃ for 1 h. After washing with PBS three times, sections were overnight incubated with rabbit anti-Gasdermin-D (GSDMD) (K009328P, Solaibio, Beijing, China) at 4 °C. After rising with PBS, the slices were incubated with goat anti-rabbit secondary antibodies (P0208, Beyotime) at room temperature for 1 h, then coated with anti-uorescence quenching sealing solution with DAPI (P0131, Beyotime) for 5 min and sealed. Terminal deoxynucleotide transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) (C1062, Beyotime) assay was performed as per the manufacturer's protocol to detect pulmonary apoptosis. Six elds with a magni cation of ×200 in 3 slices were randomly selected from each group. Under a uorescence microscope (BX53, Olympus, Tokyo, Japan), the percentage of GSDMD-positive cells and TUNEL-positive cells were calculated using the Image J (1.37v, Wayne Rasband, available through the National Institutes of Health).
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