The lipophilic cation JC-10 was used to measure the mitochondrial activity of spermatozoa (JC-10 Assay for Flow Cytometry, Sigma-Aldrich, St. Louis, MO, USA). JC-10 fluorescence changes reversibly from green (monomeric status) to orange (multimeric status) as mitochondrial membrane potential increases. Briefly, the frozen thawed semen samples were placed into polypropylene tubes at a final concentration of 1 × 106 sperm/mL. After washing with 1 mL PBS and centrifuging for 10 min at 1800 RPM, the pellet was resuspended in 500 µL of JC-10 and incubated for 1 h at 37 °C. Afterward, samples were centrifuged, resuspended in 1 mL of PBS, and analyzed by flow cytometry. Using emission filters of 535 nm and 595 nm, the two populations with green (JC-10 monomers) or orange (JC-10 aggregates) fluorescence were quantified by frequency plots. The percentage of orange-stained sperm was recorded (HMMP), representing a cell population with high mitochondrial membrane potential.
Jc 10 assay for flow cytometry
Manufactured by Merck Group
Sourced in United States
The JC-10 Assay is a laboratory instrument designed for flow cytometry applications. It is used to measure specific cellular parameters, such as mitochondrial membrane potential, in a wide range of cell types. The JC-10 Assay provides quantitative data on these cellular characteristics without making interpretations or extrapolations about its intended use.
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2 protocols using jc 10 assay for flow cytometry
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Assessing Sperm Mitochondrial Potential
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Sperm Mitochondrial Potential Evaluation
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The sperm mitochondrial status was assessed using JC-10 (lipophilic cation).
According to the manufacturer (JC-10 Assay for Flow Cytometry, Sigma-Aldrich, USA), JC-10 changes reversibly its fluorescence from green (monomeric status) to orange (multimeric status) when mitochondrial membrane potential is high. Frozen straws were thawed at 37 °C for 60 s.and the sperm suspension was collected into polypropylene tubes at a final concentration of 1 x 10 6 sperm/ml. One group of semen was induced for apoptosis using carbonyl cyanide m-chlorophenyl hydrazine (CCCP) 1 mM and incubated at 37 °C for 15 min and served as positive control. All groups were washed in 1 ml PBS by centrifugation at 600 g for 10 min, then resuspended in 500 µl of JC-10 (200x JC-10 in DMSO) and incubated 1 h at 37 °C, after that samples were centrifuged and diluted in 1 ml PBS. Flow cytometric evaluation was conducted for n=4 trials (3 rd , 4 th , 5 th and 6 th ).
According to the manufacturer (JC-10 Assay for Flow Cytometry, Sigma-Aldrich, USA), JC-10 changes reversibly its fluorescence from green (monomeric status) to orange (multimeric status) when mitochondrial membrane potential is high. Frozen straws were thawed at 37 °C for 60 s.and the sperm suspension was collected into polypropylene tubes at a final concentration of 1 x 10 6 sperm/ml. One group of semen was induced for apoptosis using carbonyl cyanide m-chlorophenyl hydrazine (CCCP) 1 mM and incubated at 37 °C for 15 min and served as positive control. All groups were washed in 1 ml PBS by centrifugation at 600 g for 10 min, then resuspended in 500 µl of JC-10 (200x JC-10 in DMSO) and incubated 1 h at 37 °C, after that samples were centrifuged and diluted in 1 ml PBS. Flow cytometric evaluation was conducted for n=4 trials (3 rd , 4 th , 5 th and 6 th ).
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