2 mercaptoethanol βme

Thymocytes were cultured at 37oC with 5% CO2 in RPMI-1640 (Gibco, Invitrogen Corporation, CA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco Invitrogen), 0.1% (v/v) 2-mercaptoethanol βME (Sigma Aldrich) and 1% (v/v) penicillin-streptomycin (Gibco Invitrogen) (RPMI-10). Recombinant TNF was supplemented to cultures at 10ng/ml, unless otherwise stated, and was obtained from R&D, with PBS used as vehicle. Inhibitors were used at the following concentrations, unless otherwise stated : specific IKK2 inhibitor BI605906 (10μM in 0.1% DMSO vehicle), pan IKK inhibitor IKK16 (2μM in 0.1% DMSO), Necrostatin 1 (1μM in 0.1% DMSO). Cells were activated by CD3 (10μg/ml) and CD28 (10μg/ml) mAb bound to 96 well flat bottom plates overnight at 4°C and washed with PBS prior to culture. Cells were cultured at 10⁶/ml in 200μl. TCR^hi CD4⁺ SP and CD8⁺ SP thymocytes were isolated by cell sorting from total thymocytes, labelled with CTV and cultured at 10⁶/ml for 72hr in either CD3+CD28 mAb coated plates or uncoated wells as control. Cultures of peripheral T cells were performed by labelling total lymph node cell suspensions with CTV and culturing cells at 10⁶/ml in 96 well flat bottom plates.

STORM imaging buffer for cultured cells contains 10% (w/v) glucose (Sigma-Aldrich), glucose oxidase (0.56 mg/ml; Sigma-Aldrich), catalase (0.17 mg/ml; Sigma-Aldrich), and 0.14 M 2-mercaptoethanol (βME, Sigma-Aldrich). For the formalin-fixed paraffin-embedded (FFPE) tissue section, to reduce the high background caused by the strong scattering of pathological tissue, an optical clearing process was conducted before imaging by immersing the sample in 60% (v/v) 2,2′-thiodiethanol (TDE) for 20 to 30 min to make the sample transparent (27 (link)). For the STORM imaging buffer of FFPE tissue section, 60% (v/v) TDE solution was used instead of water to match the tissue’s index and contains 10% (w/v) glucose (Sigma-Aldrich), glucose oxidase (0.56 mg/ml; Sigma-Aldrich), catalase (0.17 mg/ml; Sigma-Aldrich), 0.14 M βME (Sigma-Aldrich), and 0.2 mM cyclooctatetraene (Sigma-Aldrich). The imaging buffer was added to the sample dish right before imaging.

For the factor-induced neurogenic differentiation procedure, cells with 80% confluence at passages 3–5 were induced by a culture medium supplemented with 50 μM. All-trans-retinoic acid (RA; Sigma-Aldrich) and 1 mM 2-Mercaptoethanol (β-ME; Sigma-Aldrich), which were placed in a 37 °C, 5% CO₂, and 20% O₂ incubator for 24 h. Then, the differentiated cells were maintained with the DMEM containing 1% FBS for seven days [43 (link)].