Multi well fluorescent microplate reader

Intracellular ROS production was monitored using the cell-permeable chloromethyl derivative of 2′,7′-dichlorodihydrofluorescein (CM-H₂DCFDA; Thermo Fisher Scientific). K562 cells (1.0 × 10⁵/mL) were loaded with 5.0 µM CM-H₂DCFDA for 20 min in a 96-well plate. After washing with PBS, DCF fluorescence was analyzed for 120 min at λ_ex/λ_em 485/530 nm after 15 µM TR was added or with 2.5 mM EGTA plus TR in a multi-well fluorescent microplate reader (Applied Biosystems, Bedford, MA, USA). As a positive control, 2.0 mM t-BOOH was used.

Protein thiol groups (–SH) were quantified using 5,5′-dithiobis(2-nitrobenzoic) acid (DTNB, Ellman’s reagent). After 1.0 × 10⁵/mL K562 cells were incubated with 15 µM TR or 2.0 mM t-BOOH for 24 h and centrifuged for 10 min at 700× g, the cell pellet was treated with 0.2 mL of 6% trichloroacetic acid for protein precipitation, and centrifuged at 6000× g for 15 min. The resultant pellet was suspended with 1.0 mL 0.5 M PBS (pH 7.6) and after the addition of 0.1 mM DTNB, absorbance was acquired at 412 nm, and the amount of thiol groups in the control (i.e., without TR, considered to be 100%) was calculated from ε = 13,600 M⁻¹.cm⁻¹. The same procedure was applied to quantify reduced thiol groups of mitochondrial proteins isolated from those cells, with mitochondrial isolation performed as previously described [22 (link)]. Reduced glutathione (GSH) levels were fluorometrically estimated using o-phthalaldehyde [23 (link)]. An aliquot (0.1 mL) of the supernatant obtained after acid precipitation was added to 1.9 mL of a buffer containing 0.1 mg/mL NaH₂PO₄ and 5.0 mM EGTA pH 8.0, followed by the addition of 0.05 mg/mL o-phthalaldehyde. Fluorescence was analyzed at λ_ex/λ_em350/420 nm using a multi-well fluorescent microplate reader (Applied Biosystems).