Methocult gf m3534 medium

Viable bone marrow cells and splenocytes were homogeneously dispersed in the MethoCult™ GF M3534 medium (Stemcell Technologies) at densities of 1 × 10⁴/mL and 2 × 10⁴/mL, respectively. Cells were plated in 35 mm culture dishes (Stemcell Technologies) and incubated at 37°C in a humidified atmosphere of 5% CO₂. Following a 9-day (bone marrow cells) and 13-day (splenocytes) incubation, the number of CFU-GM with colonies consisting of more than 50 cells was manually counted under an inverted microscope (BX51, Olympus, Tokyo, Japan). The average colony number of the quadruplicated dishes per group was represented for each specimen.

A total of 100 LSKs were seeded in MethoCult GF-M3534 medium (StemCell Technologies) in triplicate in 35-mm plastic plates and cultured at 37°C, 5% CO₂, fully humidified air. After 7 days, colony numbers were scored.

Detection and quantification of mouse hematopoietic progenitors in peripheral blood was performed using nucleated cells isolated from the pooled peripheral blood of control or 20 mg/kg D16F7 mAb treated B6D2F1 mice (n = 4 for each experimental group) and mixed with MethoCult™ GF M3534 medium (StemCell Technologies, Vancouver, Canada). Briefly, 0.7–0.9 × 10⁶ cells were seeded in 35 mm Petri dishes (8 per group). Cells were allowed to grow for 10 days in CO₂ incubator at 37°C and colonies containing at least 30 granulocytes (CFU-G), monocytes/macrophages (CFU-M) or both cell types (CFU-GM) were counted. Results were normalized by total number of initially plated cells.

Biotin conjugated anti-mouse-CD4 (clone 34 GK1.5), anti-mouse-CD8 (clone 53-6.7), anti-mouse-CD11b (clone M1/70), anti-mouse-CD45R/B220 (clone RA3-6B2), anti-mouse-Ly6G/Gr-1 (clone RB68C5), anti-mouse-Ter119 (clone Ter119), anti-mouse-CD117 (c-kit)-APC (clone 2B8), anti-mouse -Ly-6A/EA (Sca-1)-PE (clone D7), and PERCP-conjugated streptavidin were purchased from eBioscience (San Diego, CA, USA). In addition, 2,7-dichlorodihydrofluorescein diacetate (DCFDA) was purchased from Sigma (St. Louis, MO, USA). MethoCult GF M3534 medium was purchased from Stem Cell Technologies (Vancouver, Canada). MitSox red mitochondrial superoxide indicator was obtained from Life Technologies (Grand Island, NY, USA). Rabbit anti-γH2AX was obtained from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-NOX4 was obtained from Proteintech (Wuhan, China). FITC-conjugated goat anti-rabbit antibodies were obtained from Abcam Biotechnology (Cambridge, MA, USA). Cytofix/Cytoperm buffer (554722), Perm/Wash buffer (554723), and Cytoperm Permeabilization Buffer Plus (561651) were obtained from BD Pharmingen (San Diego, CA, USA). Sitagliptin was obtained from Merck Sharp & Dohme (South Granville, NSW, Australia).