Millex gv4

Human tissues were homogenized in ice-cold phosphate-buffered saline (PBS) and centrifuged, and supernatants were collected for Hcy quantification. Hcy concentrations were determined using an Axis Homocysteine Enzyme Immunoassay Kit (Axis-Shield). To assay metabolite levels, cells were harvested by PBS washing and denatured in pre-chilled 60% methanol (in ddH₂O, pre-cooled at −80 °C for 1–2 h). Cell lysates were centrifuged (10,000 × g) at 4 °C for 5 min. Supernatants were vacuum-dried, re-dissolved in ddH₂O, and subjected to ultrafiltration on a polyvinylidene fluoride low protein binding membrane (Millex-GV4 and Millex-HV4, Millipore). Metabolites were extracted and Hcy was analyzed using LC-MS. SAM and SAH levels were detected using a SAM & SAH ELISA Combo Kit (Cell Biolabs). 1-Methylnicotinamide was measured using a UHPLC-QTOF-MS System (Agilent Technologies, 1290 LC, 6550 MS) as described previously^{99 (link)}. Each assay was repeated in triplicate, and means were used for analysis.

Intracellular homocysteine concentrations in cell cultures were measured by a Homocysteine‐EIA Kit (Bio‐Rad, Hercules, CA, USA) following the manufacturer's protocols. HTL was assayed as follows: cells were harvested by washing with PBS and then denatured using pre‐chilled 60% methanol dissolved in ddH₂O. Cell lysates were collected and centrifuged at 10,000 g for 5 min at 4°C. The supernatant was vacuum dried, redissolved in ddH₂O and then subjected to ultrafiltration on a PVDF low‐protein‐binding membrane (Millex‐GV₄ and Millex‐HV₄; Millipore, Bedford, MA, USA). The metabolites were extracted, and HTL was analysed by LC‐MS (Barathi et al, 2010).