Oci ly10

Human DLBCL cell lines OCI-Ly01, VAL, OCI-Ly10, SUDHL-8, Farage, and SUDHL-4 cells were purchased from ATCC (Shanghai, China) and cultured in RPMI-1640 medium (Hyclone, Utah, USA) supplemented with 10% fetal bovine serum (FBS) (Ausgenex, Australia), 100 U/mL penicillin, 100 U/mL streptomycin, and 2 mM L-glutamine. Human embryonic kidney 293T cells were maintained in Dulbecco modified Eagle media (DMEM; Hyclone) with 10% FBS. Cells were cultured at 37°C in a 5% CO₂ humidified incubator.
Lentiviral plasmids containing optimized CD300A short hairpin (shRNA) or scramble sequences were produced using 293T cells, and were used to infect DLBCL cells as described [4 (link), 33 (link)]. The sequences for CD300A shRNAs used in the present study included shRNA-1 (5′-GATGTTTCAGAAATGGATCAA-3′), shRNA-2 (5′-CCCAGGGAAGAACTTCACTAT-3′), and a scramble shRNA (5′-CCTAAGGTTAAGTCGCCCTCG-3′).
The surface expression of CD300A in lymphoma cells was determined using a phycoerythrin (PE)-conjugated antibody against human CD300A (clone E59.126, Beckman Coulter, Marseille, France). After staining for 30 min on ice, cells were resuspended in phosphate-buffered saline (PBS) and analyzed using a FACS Calibur instrument (Becton Dickinson, CA, USA). The isotypic antibody (clone 679.1Mc7, Beckman Coulter) was used as control.

Unless otherwise specified, cells were grown and cultured at a concentration of 1 × 10⁶ cells/mL of standard culture media (RPMI 1640 + 10% FCS, 1% penicillin/streptomycin, 1% HEPES, 1% non-essential amino acids) at 37 °C in 5% ambient CO₂. For glycosidase treatments, cultures were supplemented with kifunensine (Santa Cruz sc-201634) at a concentration of 16 ng/mL. Jurkat, Nalm6, and OCI-Ly10 parental cell lines were obtained from ATCC. Primary human T cells were purified from human peripheral blood mononuclear cells, obtained commercially from Miltenyi (Catalog #150-000-452).