To elucidate the cytotoxic profile of Mn-Oda, a wide range of biomarkers specific to apoptosis and autophagy including caspase release, mitochondrial membrane integrity, fluorogenic probe localisation, and cell cycle analysis were employed.
Hence, to confirm the mitochondrial involvement in MFX and CFX mediated apoptotic cell death, we checked mitochondrial membrane integrity using the fluorescent probe DiOC6.
To assess cellular membrane integrity, propidium iodide (PI) fluorescent probes (P4170, Sigma-Aldrich, USA) were added to the sample at a final concentration of 7 μM.
We therefore used a GST-tagged version as a probe for endosomal membrane integrity, using anti-GST antibodies to detect the bound protein.
For the assessment of plasma membrane integrity 1 μM TO-PRO-3 (Molecular Probes-Invitrogen) was used in time-lapse microscopy.
Semen samples were examined for: post-thaw motility, vigour of movement, concentration, sperm morphology, slow thermoresistance test (STT), membrane integrity, acrosome status, mitochondrial function using fluorescent probes association (FITC-PSA, PI and JC-1) and sperm chromatin integrity using acridine orange assay.
Notably, FM4 64 emission decreased after 14 min, suggesting probe (1) disrupted the outer membrane integrity over this time period.
Cell death or loss of plasma membrane integrity was determined by measuring Sytox Red (Molecular Probes–Invitrogen, Eugene, OR, USA) or propidium iodide (PI -emitted fluorescence.
A two-component fluorescent Live/Dead Baclight kit L-13152 (Molecular Probes, USA) was used to evaluate membrane integrity in nonluminescent E. coli K12 TG1 cells treated with GFNs.
Cell death was scored by Yo-Pro-1 (Molecular Probes) staining (a marker for loss of membrane integrity) and imaged by fluorescence microscopy.
