7 aad reagent

Bone marrow cells were collected from mouse femur bones by flushing the marrow cavity with PBS [21 (link),22 (link)]. Cells (2 × 10⁶ cells/mL) were centrifuged at 1500 rpm for 10 min and supernatants were removed. Anti-mouse CD16/CD32 (CD16 ‘Fc gamma III Receptor’ and CD32 ‘Fc gamma II Receptor’ are the low affinity receptors for the mouse IgG Fc portion and are expressed by B cells) antibody (1 µL) was added and incubated for 10 min on ice to block low-affinity Fc receptor expressed on other cells. Then cells were stained with 3 µL of fluorochrome-conjugated anti-mouse antibodies to CD38-Alexia (90/CD38, also known as Ab 90, CD38 is expressed at increasingly higher levels on B cells at each stage of B-cell differentiation, and is then down-regulated on germinal center B cells and mature plasma cells) and CD138-PE (Clone: 281–2, CD138 is expressed on B lymphocytes at specific stages during their differentiation). Then, the samples were incubated for 25–30 min at room temperature. After centrifugation (1500 rpm, 5 min), the cells were re-suspended in PBS and 2 µL of the 7-AAD reagent (all from BD Pharmingen) was used as a viability probe for dead cell exclusion before analysis by FACS LSR II flow cytometer (BD Biosciences). FLOW JO software (Treestar Inc., Ashland, OR) was used to analyze the data.

For cell cycle analysis, A2780 and OV90 cells were seeded in Petri dishes and treated with different concentrations of MK8722 for 48 h. Cells were washed three times with PBS, digested into cell suspensions, and then washed three times with PBS again. They were then mixed with pre-cooled 75% ethanol and stored at 4 °C for 4 h. After washing with PBS three times, 7-AAD reagent (BD Pharmingen, SD, USA) was added to the cells, and the suspension was incubated for 20 min. The DNA content of each cell cycle phase was measured. To detect apoptosis in A2780 and OV90 cells, cell suspensions obtained using the same method as mentioned above were incubated with the FITC Annexin V apoptosis detection kit (BD Pharmingen, SD, USA) for 20 min. Subsequently, the apoptosis ratio of each group was determined.