Rpmi 1640 medium without l glutamine with phenol red

THP-1 and THP-1 X-Blue™ cells were maintained in RPMI 1640 Medium without L-glutamine with phenol red (Euroclone, Pero, Italy), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Euroclone, Pero, Italy), 2 mM L-glutamine (Euroclone, Pero, Italy), and 100 U/mL penicillin/streptomicin (Euroclone, Pero, Italy). Before treatments, cells were seeded into a 96-well plate and differentiated into macrophages by 72 h incubation with 100 ng/mL phorbol 12-myristate 13-acetate (PMA, Enzo Life Sciences, New York, NY, USA) followed by 24 h incubation in RPMI medium.

Epstein–Barr Virus (EBV) immortalized PBMCs were provided by Galliera Genetic Bank, from lymphocytes of the two patients, their parents, and four healthy controls, including two children (cell lines 281 and 283) and two adults (cell lines 519 and 550). The immortalized cells were maintained in RPMI 1640 medium without L-Glutamine with phenol red (Euroclone, Milan, Italy) supplemented with 10% fetal bovine serum (FBS South America origin EU Approved, Euroclone, Milan, Italy), 1% L-Glutamine 100X 200 mM (Euroclone, Milan, Italy), and 1% penicillin–streptomycin solution 100X (Euroclone, Milan, Italy) at 37 °C in a 5% CO₂ atmosphere. Each cell line was synchronized with thymidine treatment, which requires an inoculum of 500,000 cells/mL to be cultured for 24 h in a complete medium enriched with 1% thymidine 200 mM (Merck Life Science, Milan, Italy), capable of blocking cells in the G1 phase, and for the following 24 h in complete medium without thymidine, to restart growth starting from the same stage of the cell cycle, at 37 °C in a 5% CO₂ atmosphere. At the end of the treatment, the cells were washed with HEPES 50 mM (Merck Life Science, Milan, Italy), to prevent cryo-induced pH changes, and pellets of 5 × 10⁶ cells were stored at −80 °C.