Dimethyl sulfoxide (dmso)

100μL of the cell suspension containing 2.0×10³ cells was added to each well in 96-well plate and cultured for 24 h at 37°C. After a serial dilution, adenine was added to the cell culture with the final concentrations of 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.01563, 0.00781 and 0.00391 mg/ml. The cell morphology and the number were evaluated at 0h, 24h, 48h and 72h. To evaluate the cell growth, 20μL of MTT (Amresco, Ohio, USA) at the concentration of 5mg/ml was added into each well in 96-well plate, and cells were continuously incubated for an additional 4 hours. After carefully removing the supernatant, 150μL of DMSO (Solarbio, Beijing, China) was added to each well and the plate was shaken genteelly until DMSO was completely dissolved. Optical density values of every well were measured at 570 nm by microplate reader. Inhibition ratio and 50% inhibiting concentration of adenine on the tumor cell growth were calculated. The experiments were repeated for three times.

A total of 5 × 10³ cells were added to each well of the 96-well plate and treated with 0.1625, 0.3125, 0.625, 1.25, 2.5, and 5 μM test compounds or DMSO (Beijing Solarbio Science & Technology Co., Ltd. Beijing, China) for 48 h. Then, 10 μL of MTT (5 mg/mL, in PBS) (Beijing Solarbio Science & Technology Co., Ltd. Beijing, China) or CCK-8 (Beijing Solarbio Science & Technology Co., Ltd. Beijing, China) was added into each well and further incubated for 4 h. For MTT, the samples were centrifuged at 2000 r/min for 15 min, the supernatant was removed, and 10 μL of DMSO was added to each well. Then, the cells were incubated for 10 min, and the absorbance value was measured at 490 nm using a microplate reader (Bio-Tek, Winooski, VT, USA). For CCK8, after incubation, the absorbance of the plates was measured directly at 450 nm.

We purchased DMOG (ID0540, Solarbio), KC7F2 (IK0250, Solarbio), (E)-SIS3 (SIS3) (IS1250, Solarbio), DMSO (D8371, Solarbio) from Solarbio co (Shanghai, China) and dissolve DMOG, SIS3, and KC7F2 in DMSO according to the manufacturer’s instructions. NIH-3T3 cells were inoculated in six-well plates at a density of 10,000 cells per cm² and incubation was continued for 24 h at 37 °C 5% CO₂ before intervention. According to the different groupings, NIH-3T3 cell was treated with DMOG (20 μmol/L) or KC7F2 (20 μmol/L) or SIS3 (20 μmol/L) or DMOG (20 μmol/L) + SIS3 (20 μmol/L).

FITC-labeled red blood cells were infused into bypass and sham-bypass rats (10% of blood volume) throughout the 2-h bypass period. In some experiments, the FITC-labeled red blood cells were pretreated for 3 h with either 20 μM Z-DEVD-FMK (MCE, Newark, NJ, USA) in dimethylsulfoxide (DMSO; Solarbio, Beijing, China) or the corresponding volume of DMSO, then transfused into animals during bypass. The inhibitor concentration was selected based on the literature [12 (link)]. At 2, 24 and 48 h after transfusion, blood samples were drawn from the tail vein into heparinized syringes in order to determine the percentage of stored red blood cells that had been engulfed by monocytes and neutrophils. Some rats were observed for 24 h, then they were sacrificed using sodium pentobarbital and their spleen was harvested in order to determine the percentage of stored red blood cells that had been cleared by splenic macrophages.