N acetylcystein nac

Cells were seeded in triplicates in 6-well plates containing cDMEM in numbers necessary for forming individual colonies (300 to 2500 cells depending on the cell line and condition). After 1 to 2 h attachment to the plates, cells were mock treated (DMSO 0.1%) or with temozolomide (5 µM or 10 µM, Sigma) and/or with the sphingosine kinase inhibitor SKI-II (10 µM or 20 µM; Sigma) for 2 h at 37°C, 5% CO₂. Both temozolomide and SKI-II were dissolved in DMSO and diluted in cDMEM. Cells were thereafter left non-irradiated or irradiated at different X-ray doses. After irradiation, cells were incubated for an additional 2 h in presence or absence of the drugs. At the end of this 4 h treatment, supernatants were removed by aspiration, cells were washed with PBS, fresh cDMEM was added and the plates were returned to the incubator for colony formation. Non-treated cells and non-irradiated cells were used as a control.
For cell death rescue experiments, cells were seeded, treated and incubated for colonies formation as described above, with the following modifications: 200 LN229 and 400 U87 cells per well were seeded in triplicates; 5 mM N-Acetylcystein (NAC, Sigma; dissolved in DMSO and diluted in cDMEM) was added at the time of the treatment with 20 µM SKI or 10 µM TMZ. Non-treated cells and non-irradiated cells were used as a control.

THP-1 cell line, the human acute monocytic leukemia cell line, was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). THP-1 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) in humidified atmosphere of 5% CO₂. Penicillin (50 units/ml) and streptomycin (50 μg/ml) were added to prevent bacterial contamination. Cells were maintained between 1,000 to a million cells per ml in the culture medium.
PG isolated from S. aureus, endotoxin-free bovine serum albumin (BSA), RO318220, GF109203X, LY294002, diphenyleneiodonium (DPI), N-acetylcystein (NAC), and SP600125 were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). U0126, SB202190, and Akt inhibitor IV (Akti IV) were purchased from Cell Signaling Technology (Danvers, MA, USA). Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) was purchased from Cell Sciences (Canton, MA, USA).

MN9D cells were cultivated on the poly-D-Lysine (25 µg/ml PDL: Sigma Chemical Co., St. Louis, MO, USA)-coated p-100 plate in Dulbecco's modified Eagle's medium (DMEM: Sigma) supplemented with heat inactivated 10% fetal bovine serum (FBS: BioWhittaker, Walkersville, MD, USA) in an atmosphere of 10% CO₂ at 37℃. Prior to drug treatment, culture medium was switched to serum-free, chemically-defined N2 supplement [13 (link)] containing the predetermined concentrations of D-glucose (Sigma, 5-35 mM) plus 200 µM MPP⁺. If necessary, cells were co-treated with Boc-aspartyl(OMe)-fluoromethylketone (BAF; Enzyme Systems Products, Dublin, CA, USA), N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD; Enzyme Systems Products), N-acetylcystein (NAC, Sigma), or SP600125 (Cell Signaling; Boston, MA, USA).