After segregating 2 million sperms from the samples, a buffer containing TNE/NaCl/EDTA was added to the vessel to increase the volume to 1 mL. In the case tube, 400 μl acid-detergent solution was added to 200 μl of the attenuated semen sample and was later mixed with 1200 μl of acridine orange staining solution (Sigma, St. Louis, USA), however, in the control tube the first step was bypassed. Next, the level of DNA fragmentation (percentage) was evaluated using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). An approximate number of 10,000 spermatozoa/sample were analyzed.
Acridine orange staining solution
Manufactured by Merck Group
Sourced in United States
Acridine orange staining solution is a laboratory reagent used in microscopy and flow cytometry applications. It is a fluorescent dye that binds to nucleic acids, allowing for the detection and visualization of cells, tissues, or other biological samples. The solution provides a simple and reliable method for staining and differentiating between live and dead cells, as well as for analyzing DNA and RNA content.
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Lab products found in correlation
Facscalibur flow cytometer, by BD (1 mentions)
Vectashield antifade mounting media, by Vector Laboratories (1 mentions)
Polyvinylidene fluoride pvdf membrane, by Merck Group (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
3 methyladenine 3 ma, by Merck Group (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by GE Healthcare (1 mentions)
Fisetin, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
6 protocols using acridine orange staining solution
1
Sperm DNA Fragmentation Analysis
2
Sperm DNA Fragmentation Assay
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As originally described in Evenson et al., [13 (link)], 2 × 106 spermatozoa per sample were resuspended in TNE 1× solution to obtain a final volume of 1 ml. Then, 1.2 ml of acridine orange staining solution (Sigma, St. Louis, USA) was mixed with 200 μl of sample (controls) or 400 μl of acid–detergent solution and 200 μl of sample brought together for 30 s (tests). At least 10,000 sperm cells were evaluated to determine the percentage of DNA fragmentation.
3
Acridine Orange Staining Protocol
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Sections were incubated in 0.1 M HCl at room temperature for 1 min. Slides were then incubated in acridine orange staining solution (Sigma, Gillingham, UK) for up to 5 min followed by a quick rinse in PBS. Sections were mounted with VECTASHIELD® Antifade Mounting Media (Vector laboratories, Burlingame, CA, USA).
4
Sperm DNA Integrity Evaluation by SCSA
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Sperm chromatin structure assay (SCSA) is a flow
cytometry technique, which measures the susceptibility
of sperm DNA to acid-induced DNA denaturation in situ.
Briefly, frozen semen samples were quickly liquefied in
a water bath at 37˚C and diluted to a concentration of
1-2×106 sperm per milliliter. Then, 200 μL of the obtained
suspension was treated with 200 μL acid-detergent
solution for 30 seconds. Afterwards, 900 μL of acridine
orange (AO) staining solution (Sigma-Aldrich, St. Louis,
MO, USA) was added, and the sample was analyzed by
flow cytometry. For each sample, a total of 10,000 events
were measured at a flow rate of approximately 200 cells/
sec. DNA fragmentation index (DFI) as a measurement
of a degree of sperm DNA damage was identified by the
reflection of red fluorescence to total fluorescence and
DFI values, subsequently analyzed using the FlowJo
software (19 (link)).
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Sperm chromatin structure assay (SCSA) is a flow
cytometry technique, which measures the susceptibility
of sperm DNA to acid-induced DNA denaturation in situ.
Briefly, frozen semen samples were quickly liquefied in
a water bath at 37˚C and diluted to a concentration of
1-2×106 sperm per milliliter. Then, 200 μL of the obtained
suspension was treated with 200 μL acid-detergent
solution for 30 seconds. Afterwards, 900 μL of acridine
orange (AO) staining solution (Sigma-Aldrich, St. Louis,
MO, USA) was added, and the sample was analyzed by
flow cytometry. For each sample, a total of 10,000 events
were measured at a flow rate of approximately 200 cells/
sec. DNA fragmentation index (DFI) as a measurement
of a degree of sperm DNA damage was identified by the
reflection of red fluorescence to total fluorescence and
DFI values, subsequently analyzed using the FlowJo
software (19 (link)).
5
Fisetin-Induced Autophagy in Cancer Cells
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Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (DMEM/F-12), fetal bovine serum (FBS), and trypsin-EDTA were purchased from GE Healthcare Life Sciences (Hyclone, Logan, UT, USA). Fisetin, dimethyl sulfoxide (DMSO), methylthiazolyldiphenyl-tetrazolium bromide (MTT), acridine orange (AO) staining solution, and 3-methyladenine (3-MA) were purchased from Sigma (St. Louis, MO, USA). RIPA Lysis and Extraction Buffer was purchased from Thermo Fisher Scientific (San Jose, CA, USA). Bradford protein assay was purchased from Bio-Rad (Richmond, CA, USA). Polyvinylidene fluoride (PVDF) membranes were purchased from Millipore (Billerica, MA, USA). SuperSignal West Femto was purchased from Pierce (Rockford, IL, USA). All other chemicals and reagents were purchased from Sigma unless otherwise specified.
6
Sperm Chromatin Structure Assay (SCSA) Protocol
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The SCSA® was conducted in accordance with the recommendations of its developer [36 (link)]. After evaluation of the sperm concentration, 2. 106 sperm cells were suspended in TNE buffer (50 mM Tris HCl pH 7.4, 100 mM NaCl, 0.1 mM EDTA; Merck, Darmstadt, Germany) to a final volume of 1 ml. The SCSA was performed on 1/5 aliquot of the spermatozoa suspension to which 400 μl of acid-detergent solution was added. Then 1.2 ml acridine orange (AO) staining solution (Sigma, St. Louis, USA) was added for 30 s. The samples were analyzed with a FAX-Calibur cytometer (BD Biosciences, San Jose, CA, USA). At least 10,000 sperm cells were counted and the results were presented in the conventional manner with the DFI (DNA Fragmentation Index %) and HDS (High DNA Stainability %) scores.
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