L glutamine

Neuronal cells (20,000) were plated in each well of Matrigel-treated 24-well plates and were cultured in DMEM/F12 medium supplemented with 2% serum replacement (“Gibco”, Carlsbad, CA, USA), 1 mM non-essential amino acids (“Paneco”, Moscow, Russia), 2 mM L-glutamine (“ICN Biomedicals Inc.”, Hackensack, NJ, USA), penicillin–streptomycin (50 U/mL; 50 µg/mL) (“Paneco”, Moscow, Russia), 1% B27 supplement (“Life Technologies”, Carlsbad, CA, USA), 5 µM forskolin (“Stemgent”, Cambridge, Massachusetts, USA), 20 ng/mL BDNF, 20 ng/mL GDNF and 200 µM ascorbic acid (all from “Peprotech”, Cranbury, NJ, USA) in a CO₂ incubator at 5% CO₂ and 37 °C. On the day of the experiment, the growth medium was changed to DMEM/F12 medium supplemented with 2 mM L-glutamine (“ICN Biomedicals Inc.”, Hackensack, NJ, USA) and penicillin–streptomycin (50 U/mL; 50 µg/mL) (“Paneco”, Moscow, Russia). N-ADA or N-DDA (5 µM) were added to the cells at the same time point and the control cultures were treated with an equal volume of DMSO. Cells were then incubated for 40 min and treated with 200 µM H₂O₂; the same volume of growth medium was used for the control wells. At 3 and 24 h time points of incubation, the cells were collected for RNA extraction and preparation of cell lysates and the conditioned growth media were collected for ELISA.

HeLa 229 cells (ABAC, Buenos Aires, Argentina) were cultured in high glucose Dulbecco’s modified Eagle’s medium (GIBCO BRL, Buenos Aires, Argentina) supplemented with 10% (v/v) fetal bovine serum (Internegocios SA, Buenos Aires, Argentina), 0.3 mg/mL L-glutamine (ICN Biomedicals Inc.), and 1.55 mg/mL glucose (Biopack) without antibiotics in 5% CO₂ at 37 °C. Chlamydia trachomatis serotype L2 (CT) were gently given by Unidad de Estudios de Chlamydia (Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina. For infection experiments, cell and bacteria were centrifuged 15 min at 1000× g. After 2 h, the medium was refreshed and cells were cultured for the indicated post-infection times (pi). Bacteria were used at a multiplicity of infection (MOI) of 1.

Bacteria used were Chlamydia trachomatis serovar L2 434/Bu (Ct) (gently given and typified by Unidad de Estudios de Clamidias, UBA, Bs. As., Argentina); serovar D (generously provided by Instituto Malbran, Bs. As., Argentina); and, a green fluorescent Ct L2 strain harboring p2TK2-SW2 IncDProm-RSGFP-IncDTerm serovar L2 (GFP-Ct) kindly provided by Derré^{50 (link)}. Bacteria were propagated in HeLa 229 or McCoy cells, harvested, purified, and quantified as previously described by Del Balzo et al.⁵¹ . HeLa cells (ABAC, Argentina) were cultured in high glucose Dulbecco´s Modified Eagle´s Medium (GIBCO, Thermo Fisher Scientific, Argentina) supplemented with 10% (v/v) fetal bovine serum (FBS) (Internegocios SA, Argentina), 0.3 mg/mL l-glutamine (ICN Biomedicals Inc) and 1.55 mg/mL glucose (Biopack, Argentina) without antibiotics in 5% CO2 at 37 °C.