ROS generation by the sperm mitochondria was detected by flow cytometry using MitoSOX™ Red dye (MSR; Molecular Probes). This reagent was prepared at a dilution of 1 µL of 5 mM MSR in 249 µL BWW. Sytox® Green dye (SyG, Molecular Probes) was then added at a dilution of 1 µL 12.5 mM SyG in 249 µL BWW in order to differentiate live and dead cells (required because trace amounts of ethidium in the MSR probe can bind directly to the nuclei of dead cells with compromised plasma membrane integrity). As a positive control, arachidonic acid (AA; Sigma Aldrich) was added at a dilution of 1 µL 50 mM AA in 99 µL BWW because this fatty acid is known to stimulate mitochondrial ROS production by spermatozoa (Aitken et al. 2013 (link)); this positive control generated an average ± s.e. response in 91.3 ± 1.7% of cells. Once stained with MSR and SyG solutions, the spermatozoa were incubated at 37°C for 15 min before centrifugation and resuspension in BWW. The results were ultimately expressed as the percentage of live MSR-positive cells/all cells and percentage of live MSR-positive cells/live cells.
Arachidonic acid aa
Manufactured by Merck Group
Sourced in China, Hungary, United States
Arachidonic acid (AA) is a polyunsaturated fatty acid that serves as a core structural component of cell membranes. It is essential for various physiological processes within the human body.
Automatically generated - may contain errors
Lab products found in correlation
Mitosox red dye, by Thermo Fisher Scientific (1 mentions)
Eicosapentaenoic acid, by Merck Group (1 mentions)
Du145, by Shanghai Cell Bank (1 mentions)
Adenosine diphosphate adp, by Merck Group (1 mentions)
Antisedan, by Pfizer (1 mentions)
Adenosine diphosphate (adp), by Merck Group (1 mentions)
Gw6471, by Merck Group (1 mentions)
Am630, by MedChemExpress (1 mentions)
Jzl184, by Selleck Chemicals (1 mentions)
Am 251, by MedChemExpress (1 mentions)
Wwl70, by Merck Group (1 mentions)
T0070907, by Selleck Chemicals (1 mentions)
6 protocols using arachidonic acid aa
1
Sperm Mitochondrial ROS Detection
2
Culturing DU145 Prostate Cancer Cells
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The human prostate carcinoma cell line DU145 was purchased from the Shanghai cell bank of Chinese Academy of Sciences (Shanghai, China). DU145 cells were cultured in Dulbecco’s modified eagle medium (DMEM, Gibico, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Hangzhou Sijiqing Company, China), 100 U/mL penicillin and 0.1 mg/mL streptomycin. Cells were incubated in a 5% CO2 incubator (Sanyo Electric Biomedical CO., LTD., Japan) at 37 °C. The media were refreshed every 2–3 days. Fish oil (Shanghai Haling biotechnology Co., Ltd., Shanghai, China), DHA, eicosapentaenoic acid (EPA), and arachidonic acid (AA, 50 mmol/L, Sigma) were prepared using NaOH and were dissolved in BSA (0.01 mol/L PBS, 1:9). Media with and without BSA were used as BSA and blank controls.
3
Platelet Aggregation Assay Protocol
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Platelet function was measured by Born light transmission aggregometry (Carat TX4 Aggregometer, Carat Diagnostics, Budapest, Hungary) at enrolment (84–96 h after hospital admission) and at 3-month and 12-month follow up. Platelet rich and poor plasma were separated from citrate-anticoagulated blood by centrifugation (180 g and 1800 g for 10 min, respectively). Platelet agonists were 1 and 2 μg/ml collagen (COLL, Takeda, Austria), 2.0 μg/ml epinephrine (EPI, Richter-Gedeon Ltd., Hungary), 0.5 μg/ml arachidonic-acid (AA, Sigma-Aldrich Ltd., Hungary) and to assess effectiveness of P2Y12 receptor inhibition 1.25, 5 and 10 μM adenosine diphosphate (ADP, Sigma-Aldrich Ltd., Hungary). In order to assess the effect of full in vitro P2Y12 and alpha-2 adrenergic platelet receptor inhibition, platelet rich plasma samples (300 μl) were preincubated with antagonists for 30 s; cangrelor, a specific, competitive platelet P2Y12 receptor antagonist (1 μM, The Medicines Company, Parsippany, NJ, USA) and atipemazol, a specific alpha-2 adrenergic receptor antagonist (2 μM, Antisedan, Pfizer Animal Health, Extron, PA, USA) were used. Measurement time was 7 min with 1,000 /min stirring at 37°C; maximal aggregation was determined in percentage.
4
Platelet Aggregation Assay Protocol
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Venous blood anticoagulated with citric acid was obtained from our laboratory members who were healthy and had not taken any medication affecting platelet function in the previous two weeks. Platelet-rich-plasma (PRP) was prepared by centrifugation of blood at 1000 rpm × 10 min at room temperature and transferred to polypropylene tubes. To induce platelet aggregation, platelets were activated in the absence or presence of agonists. Particularly, aliquots (85 μL) of PRP were preincubated with 100 μM NBP, OHNBP or Asp for 10 min. For platelet labeling, 1 μL of anti-human CD61-FITC was added for 15 min at room temperature in the dark. The agonist was then added for 5 min at 37 °C under low shear stress conditions. The following platelet agonists were used: 1 mM arachidonic acid (AA, #10931, Sigma, St. Louis, MO, USA), 10 μg/mL collagen (#AG005K, Shanghai Guandong Biology, Shanghai, China) and 10 μM ADP (#A5285, Sigma, St. Louis, MO, USA). After incubation, samples were fixed with 400 μL of 4% PFA. Finally, platelet aggregates were analyzed by FCM as CD61-FITC positive events in the region.
5
Vinegar Bioactive Compound Profiling
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RV from different aging time (1, 4, 5, 7, 14, 20, 30 months) and five batches of WV were collected. The content of TMPZ in different vinegars was determined by HPLC method (Additional file 1 : Table S1, Figure S1) [15 ]. Ion exchange resin (UBK530, WK40, 731, WA30, SK1B) were obtained from Beijing green grass bouquet technology development Co. Ltd. ADP was from Beijing Biotopped Science & Technology Co., Ltd. Arachidonic acid (AA) was purchased from Sigma (St. Louis, MO). TT, PT, APTT, FIB kit was from Beijing Steellex Instrument CO.
6
Maintaining NSCLC Cell Culture
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To maintain NSCLC cancer cells, RPMI 1640 medium containing 10% (v/v) fetal bovine serum (FBS) was applied. A hemocytometer was used to trypsinize and count cells. A total of 24 h after plating, serum-free medium took place of the complete medium for 24 h. Cells were cultivated in the medium named serum-free RPMI 1640 with JZL184 (Selleck) (1 µM, 8 h), WWL70 (Sigma) (10 µM, 8 h), KT203 (APExBIO)(10 nM, 8 h), AM251 (Medchemexpress) (50 nM, 8 h), AM630 (Medchemexpress) (50 nM, 8 h), GW6471 (Sigma) (1 µM, 8 h), T0070907 (Selleck) (1 µM, 8 h), Arachidonic acid (AA) (Sigma) (50 µM, 8 h), C16:0 MAG (5 nM, 10 nM, 100 nM, 1 µM, 5 µM, 10 µM, 8 h, add FA-free BSA to give a final concentration of 1%), C18:1 MAG (5 nM, 10 nM, 100 nM, 1 µM, 5 µM, 10 µM, 8 h, add FA-free BSA to give a final concentration of 1%), C16:0 FA (5 nM, 10 nM, 100 nM, 1 µM, 5 µM, 8 h), C18:1 FA (5 nM, 10 nM, 100 nM, 1 µM, 5 µM, 8 h) or vehicle (DMSO) at 0.1%. For cells of shControl or shABHD6 groups, the medication time was same. Cells were harvested for migration and invasion or other further assays. To avoid lipotoxicity from FA overload, we examined whether BSA supplementation affect our migration study or not. The conjugation of FAs to 1% BSA did not show additional effect on cell migration.
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