Blood cell lysis buffer

Pelvic and femoral bones were collected from 8- to 10-week-old mice and sterilized in 70% EtOH. The bone ends were cut, and the BM was flushed out using PBS. Following treatment with blood cell lysis buffer (BioLegend), the cell pellet was resuspended and cultured for 5 days in L929-conditioned media, with media changed every 2 days. After 5 days, BMDMs were polarized toward M1 phenotype by IFN-γ (200 ng/mL) (BioLegend) treatment or M2 phenotype by IL-4 (25 ng/mL) (PeproTech) treatment for 24 hours.

Spleens, collected from 8- to 12-week-old mice under aseptic conditions, were mechanistically disrupted through a 70 μm cell strainer into a single-cell suspension. Following treatment with blood cell lysis buffer (BioLegend), CD8⁺ and CD4⁺ T cells were isolated using magnetic cell sorting by negative selection (Pan T cell Isolation Kit II, Miltenyi Biotec) according to the manufacturer’s instruction. T cells were then plated in plates coated with anti-CD3 (0.5 μg/mL, clone 2C11, Bio X Cell) and anti-CD28 (5 μg/mL, clone 37.51, Bio X Cell) antibodies in RPMI 1640 medium supplemented with 10% FBS, L-glutamine (2 mM), and 2-mercaptoethanol (50 μM). After 24 hours, IL-2 (50 U/mL, PeproTech) was added to the medium. T cells were then allowed to proliferate for an additional 48 hours before treatment with MNK inhibitors. After 48 hours, T cells were collected and analyzed for the expression of CD69, PD-1, and Prf1 by flow cytometry.