The ratio of the decrease in the cell mitochondrial membrane potential and cell apoptosis after nanoparticle treatment was analysed by flow cytometry. H9C2 cells (1×106 cells well−1) were seeded in 6‐well plates and cultured for 24 h at 37 °C. Nanoparticles (30 µg mL−1) were added to 6‐well plates, and the cells were subjected to OGD/R treatment. After treatment, all the cells were collected, and the cells were stained according to the instructions of the apoptosis detection kit (331 200, Thermo Fisher Scientific) and MMP detection kit (V35116, Thermo Fisher Scientific). Then, the stained cells were analyzed by flow cytometry (Cytoflex, Beckman, CA, USA).
Apoptosis detection kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Apoptosis detection kit is a laboratory tool designed to detect and analyze apoptosis, a form of programmed cell death. The kit provides the necessary reagents and protocols to identify and quantify apoptotic cells through various techniques, such as flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Facscalibur, by BD (11 mentions)
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Streptozotocin (stz), by Merck Group (2 mentions)
Gapdh, by Abcam (2 mentions)
Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Annexin 5 fitc, by Thermo Fisher Scientific (2 mentions)
Accuri c6 flow cytometer, by BD (2 mentions)
Facscalibur system, by BD (2 mentions)
Trypan blue staining solution, by Thermo Fisher Scientific (2 mentions)
Facscalibur cytometer, by BD (2 mentions)
Facs caliber, by BD (2 mentions)
Brdu flow kit, by BD (2 mentions)
Tcs sp5, by Leica (1 mentions)
C jun, by Abcam (1 mentions)
Dynamo flash sybr green qpcr kit, by Thermo Fisher Scientific (1 mentions)
Fosl1, by Abcam (1 mentions)
P erk, by Abcam (1 mentions)
C fos, by Abcam (1 mentions)
Prestained protein marker, by Thermo Fisher Scientific (1 mentions)
78 protocols using apoptosis detection kit
1
Nanoparticle-Induced Mitochondrial and Apoptotic Changes
2
TUNEL Analysis of Apoptosis in Mouse Substantia Nigra
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The substantia nigra (SN) region of the mouse brain was cut at 40 μm on a cryostat and stored at −80 °C. To perform terminal transferase-mediated dUTP nick end-labeling (TUNEL) analyses, sections were fixed in 4% PFA for 30minutes and then washed several times with room temperature PBS. Then, the sections were incubated in cold ethanol/acetic acid 2:1 for 5mins and washed in PBS again. The labeling of neuronal apoptosis in SN sections was performed using the apoptosis detection kit purchased from ThermoScientific (USA), which is based on the in situ TUNEL technique using terminal deoxynucleotidyl transferase (TdT) and the images though confocal microscope were acquired (Leica TCS SP5).
3
Apoptosis Analysis of Drug-Treated Cells
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The cells were seeded in a 10-cm culture dish. After 3 days of drug treatment, the cells were resuspended in 200 μL of binding buffer, and the apoptosis assay was then performed with the Apoptosis Detection Kit (Thermo Fisher Scientific, Inc., Wilmington, DE, USA). The flow cytometer was used to analyze the apoptosis results.
4
FOSL1 Knockdown Modulates Diabetic Progression
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Short hairpin (sh) RNA targeting FOSL1 (sh-FOSL1), sh-negative control (sh-NC), adenovirus vector (Ad)-sh-FOSL1, and Ad-sh-NC were purchased from Sangon Biotech (Shanghai, China). Streptozotocin (STZ) was obtained from Sigma Aldrich (San Luis, MO, USA). The RevertAid H Minus First Strand cDNA Synthesis kit, DyNAmo Flash SYBR Green qPCR kit, and apoptosis detection kit were all purchased from Thermo Fisher Scientific (Waltham, MA, USA). The primary antibodies (FOSL1, p-ERK, ERK, c-fos, c-jun, and GAPDH) and the HRP-conjugated secondary antibody used for western blotting were procured from Abcam (Cambridge, UK).
5
Establishing Cell Culture Conditions
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Fetal bovine serum (FBS) was obtained from Gemini Bio (California, USA). RPMI 1640 medium, ECL-PLUS/Kit, prestained protein marker, apoptosis detection kit, and puromycin were purchased from Thermo Fisher Scientific (MA, USA). TRIzol, trypsin, dNTPs, oligo dT, Rnase Inhibitor were obtained from Invitrogen (Carlsbad, CA, USA). trypsin, BCA protein assay kit and RIPA lysis buffer were obtained from Beyotime biotechnology (Shanghai, China). GIEMSA staining reagent and paraformaldehyde were purchased from Dingguo Biotechnology (Shanghai, China).
6
Apoptosis and Proliferation Analysis of BMSCs
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Cells were stained using an apoptosis detection kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instructions 31 (link). Briefly, BMSCs were harvested after cultured with different conditioned media or culture medium (Ctrl) supplemented with 10% FBS for 3 days. The BMSCs were then resuspended in 1× annexin-binding buffer (100 μL) and incubated with 1 μL propidium iodide (PI, 100 μg/mL) and 5 μL Annexin V for 30 min in the dark and analyzed via flow cytometry.
Cell proliferation was monitored using Cell Counting Kit-8 (CCK-8) (n = 6). Briefly, BMSCs were seeded in 96-well plates at a density of 3×103 cells/well and cultured in conditioned media or control medium supplemented with 10% FBS for 1, 3, 5 and 7 days.
Cell proliferation was monitored using Cell Counting Kit-8 (CCK-8) (n = 6). Briefly, BMSCs were seeded in 96-well plates at a density of 3×103 cells/well and cultured in conditioned media or control medium supplemented with 10% FBS for 1, 3, 5 and 7 days.
7
LO2 Cell Apoptosis and ROS Assay
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LO2 cells apoptosis was assessed by flow cytometric analysis. Briefly, the collected LO2 cells (1 × 105 cells/mL) were cultured in 96-well plates for 24 h, and then were stained with V-FITC and PI using an apoptosis detection kit (Thermo Fisher Scientific; cat. no. 88–8005-74) at 25°C for 20 min in the dark. The apoptotic cells were measured using a flow cytometer (BD Biosciences).
Meanwhile, flow cytometry analysis was also used for the ROS generation assay. Briefly, LO2 cells were stained with DCFH-DA (Sigma Aldrich; cat.no. D6883; 1µM) and incubated for 30 min in the dark at 37°C. After that, the stained cells were washed with PBS and the cell populations were analyzed using a flow cytometer.
Meanwhile, flow cytometry analysis was also used for the ROS generation assay. Briefly, LO2 cells were stained with DCFH-DA (Sigma Aldrich; cat.no. D6883; 1µM) and incubated for 30 min in the dark at 37°C. After that, the stained cells were washed with PBS and the cell populations were analyzed using a flow cytometer.
8
Sirtuin-1 Regulation of Diabetic Retinopathy
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Angio-proteomie (Boston, MA, USA) provided human retinal endothelial cells (HRECs). Cell culture and transfection-related reagents were from Invitrogen (Carlsbad, CA, USA). SIRT1-siRNA (si-SIRT1-1/-2) and the negative control (si-NC) were from Sangon Biotech (Shanghai, China). The commercial kits for the measurement of glucose and triglyceride contents were from Boxbio (Beijing, China). The caspase-3 assay kit was from QCbio Science & Technologies (Shanghai, China). Sigma Aldrich (San Luis, MO, USA) provided streptozotocin (STZ) and the commercial kits for the measurement of reactive oxygen species (ROS), catalase (CAT), superoxide dismutase (SOD), and inflammatory cytokines. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-h-tetrazolium bromide (MTT) was from Aladdin (Shanghai, China). Apoptosis detection kit and western blotting analysis-related reagents were from Thermo Fisher Scientific (Waltham, MA, USA). Rat VEGF ELISA kit, the primary antibodies (Notch1, Hes1, Hey2, SIRT1, and GAPDH), and the HRP-conjugated secondary antibody were procured from Abcam (Cambridge, UK).
9
Neurodegeneration Staining and Apoptosis Detection
10
Apoptosis Analysis of Treated Cells
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Total viable cells were assessed on a Neubauer hemacytometer (Hausser Scientific, Horsham, PA, USA) using the trypan blue (Life Technologies) exclusion method. Apoptosis analysis was performed using an Apoptosis Detection kit (Thermo Fisher Scientific). After being treated with inhibitors for 24 or 48 h, the cells were pelleted and resuspended in binding buffer with PI and APC-conjugated Annexin V at room temperature for 15 min. Cells were analyzed using a FACS Calibur (BD Bioscience, San Jose, CA, USA). Total apoptotic cell numbers were calculated as the sum of ‘early’ apoptotic cells (Annexin V+ only) and ‘late’ apoptotic cells (Annexin V+/PI+).
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