Apoptosis is energy consuming, and therefore depends on minimal residual ATP concentrations, whereas necrosis is the default route in the absence of ATP [39]. Induction of apoptosis generally follows one of two pathways. serum-free DMEM, and resuspended in serum free DMEM comprising annexin V (1:40) for 30?min in the dark at 37C inside a humidified 5% CO2/95% air flow atmosphere. Cells were subsequently washed and resuspended in serum free DMEM comprising PI (1:40). Cells were measured having a FACSCalibur (Becton Dickinson, San Jose, CA, USA). Results were analyzed by Cell Pursuit Pro software (Becton Dickinson). Detection of caspase-3 activity Cells were grown inside a 96?wells plate (20,000 cells/well). After treatment with Hcy and/or Z-VAD FMK, cells were lysed and incubated with DEVD-rhodamine 110 substrate (Roche, Mannheim, Germany) for 1?h at 37C. Subsequently the amount of free rhodamine was identified at a microplate fluorescence reader (TECAN spectrafluor, Switzerland). The developed fluorochrome was proportional to the concentration of activated caspase-3 and could be quantified by a calibration curve of diluted free rhodamine. Each condition Alendronate sodium hydrate was measured in triplo per measurement (total of three measurements). Immunofluoresence microscopy To measure the manifestation of NOX2 and the putative formation of nitrotyrosin, cells were incubated with or without Hcy for Alendronate sodium hydrate 24?h in the 4-well chamber slides (Nalge Nunc International, Naperville, IL, USA). Cells were washed with PBS and fixated with 4% formaldehyde for 10?min at 37C. Cells were consequently washed with PBS, permeabilized with acetoneCmethanol (70%C30%) for 10?min at ?20C, and then washed again with PBS/Tween-20 (0.05% (v/v) Tween-20 in PBS). Subsequently cells were incubated with main antibodies against NOX2 and nitrotyrosin for 60? min at space temp followed by incubation over night at 4C. PBS and isotype settings were also tested to determine nonspecific binding of the antibodies and background transmission. The following day time the cells were washed with PBS/Tween and incubated with the secondary antibodies for 30?min at room temperature. After subsequent washes in PBS/Tween and PBS, the slides were covered in mounting medium comprising DAPI (Vector Laboratories Inc, Burlingame, CA, USA) to visualize nuclei. Thereafter the slides were covered with coverslips. Subsequently, cells were analyzed by means of a 3I MarianasTM digital imaging microscopy workstation (Zeiss Axiovert 200?M inverted microscope; Carl Zeiss, Sliedrecht, Netherlands), equipped with a nanostepper engine (for 5?min. Cells were then counted and equivalent amounts were taken per condition. After centrifugation for 2?min (400value (two sided) of 0.05 or less was considered to be significant. Results Measurement of the concentration of d,l-homocysteine (d,l-Hcy), l-homocysteine (l-Hcy), S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH) We tested the effects of d,l-Hcy at concentrations of 0.1?mM, 1.1?mM and 2.7?mM, in accordance with previous studies in isolated vascular cells [15, 17, 26, 29C31]. We quantified the exact concentration of d,l-Hcy in medium Alendronate sodium hydrate added to the cells, via HPLC. As depicted in Table?1, the concentration of d,l-Hcy was according to the concentration added to the growth medium. Table?1 Concentration of d,l-Hcy and l-Hcy in culture medium on P /em ?=?0.006); but an increase in S-adenosylhomocysteine (SAH, *** em ?P /em ? ?0.001) was detected, compared to all other concentrations Since earlier studies have shown that only the l form SAV1 of Hcy is bioactive [25, 26], we also determined the l-Hcy concentration in medium by IMx. We found that 42.7%, 42.5% and 43.3% of the total d,l-Hcy amount 0.1?mM, 1.1?mM and 2.7?mM, respectively, in fact was l-Hcy at em t /em ?=?0. Furthermore, we identified the concentrations of l-Hcy at 24?h, the final time point of incubation, and found out a significant decrease in the concentrations of l-Hcy, namely for 1.1?mM a significant decrease of 0.14?mM l-Hcy; +/?0.011 ( em P /em ? ?0.001) and for 2.7?mM a significant decrease of 0.34?mM l-Hcy; +/?0.0301 ( em P /em ? ?0.001). It has Alendronate sodium hydrate been well recorded that increased levels of SAH inhibit several important methylation reactions [32C34]. Consequently we also identified the intracellular concentrations of both SAH and SAM, being one of the main methyl donors (Table?1). Measuring intracellular concentrations of SAM and SAH in H9c2 cells incubated with and without d,l-Hcy showed a significant depletion of SAM at 2.7?mM d,l-Hcy compared to untreated cells and 0.1?mM d,l-Hcy treated cells ( em P /em ?=?0.006; em n /em ?=?3). While the concentration of SAH increased significantly at this same concentration compared to all other conditions ( em P /em ? ?0.001; em n /em ?=?3). Consequently we also examined the effects of the higher, non-physiological concentrations of Hcy. Effect of Hcy on cell viability We analyzed the effect of Hcy on H9c2 cell viability by measuring annexin V and/or PI positivity using flow-cytometry. Incubation with 0.1?mM d,l-Hcy had no effect on single-annexin-V-positivity (Fig.?1A), double-annexin-V, PI positivity (Fig.?1B) or single-PI positivity (not shown, approximately 3% for each condition). In contrast, incubation with 1.1?mM d,l-Hcy resulted in a significant increase in solitary- annexin-V-positive cells ( em P /em ? ?0.002) (Fig.?1A). This increase in single-annexin-V-positive.