L glutamine

Doctor Chiara Baldo of the Laboratorio di Genetica Umana, IRCCS Istituto Giannina Gaslini, Genoa, performed the EBV immortalization. LCLs with mutations in the RNASEH2A (p.R108W + p.F230L) and RNASEH2B (p.A177T) genes were studied, as well as one healthy control (CTRL). Cell lines were grown in RPMI 1640 medium (CARLO ERBA Reagents S.r.l., Cornaredo, Italy), supplemented with 20% fetal bovine serum (FBS) (CARLO ERBA Reagents S.r.l., Cornaredo, Italy), 0.3 mg/L L-glutamine, and 5% penicillin-streptomycin (CARLO ERBA Reagents S.r.l., Cornaredo, Italy) at 37 °C in a humidified atmosphere with 5% of CO₂. Centrifugation was used to pellet cells, which were then washed in 1X PBS and treated as needed.

Human dermal fibroblasts from healthy adult donors (aged 35–45) were obtained from the “Cell Line and DNA Biobank from Patients Affected by Genetic Diseases—NETWORK OF GENETIC BIOBANKS TELETHON” [43 ]. Fibroblasts were used for the experiments between passages 11 and 13. The experiments were performed using fibroblasts from two different donors.
Fibroblasts were grown in RPMI-1640 (Carlo Erba Reagents, Milan, Italy) supplemented with 10% Fetal Bovine Serum (FBS, Carlo Erba Reagents, Milan, Italy) and 2 mM L-Glutamine (Carlo Erba Reagents, Milan, Italy). Cultures were incubated at 37 °C in 95% humidity and 5% CO₂ atmosphere. At confluence, fibroblasts were detached from culture flasks by treatment with trypsin (Carlo Erba Reagents, Milan, Italy). Cells were thawed on an average of two weeks before each experiment to have sufficient numbers of cells. Cells were routinely checked for the presence of mycoplasma by using the nested PCR method described in Tang et al. [44 (link)].