Particle count and size analyzer

For cell proliferation analysis, Karpas 299 and KMS-18 cells were electroporated with the indicated siRNAs. Twenty-four hours after transfection, cells were placed at 1.7 × 10⁵ cells/mL (Karpas 299) and 0.5 × 10⁶ cells/mL (KMS-18) per well in 6-well plates in triplicate. Cells were counted at 24-hour intervals for a total of 72 hours or 96 hours using a Z2 Beckman Coulter Particle Count and Size analyzer. Quantitative PCR and cell cycle analysis were performed as previously described [53 (link)]. To assess protein half-life, cycloheximide (Sigma) was added to cells (electroporated with the indicated siRNAs) at 100 μg/mL 60 hours post-transfection. Cells were harvested at the indicated time points and analyzed by immunoblotting.

MCF-7 and MDA-MB-231 cells were seeded in 6-well culture plates (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ, USA) at a concentration of 1 × 10⁶ cells/well in a complete medium. The following day, the cells were refed with DMEM supplemented with 0.1% FBS containing 0.1 mg/mL 5-FU, 0.1 mg/mL 5-FU + 0.3 µg/mL F6 and 0.3 µg/mL F6. The plates were incubated for 24 h at 37 °C in an atmosphere of 5% CO₂. Then, the cells were trypsinized and centrifuged, and cell pellets were resuspended in phosphate-buffered saline (PBS). Cell count was performed by a particle count and size analyzer (Beckman Coulter Inc., Fullerton, CA, USA). Three replicate wells were used for each data point, and the experiment was performed six times.

For proliferation assay, cells were seeded in 35 mm Petri dish at a concentration of 5 × 10⁴ cells/dish in a complete medium. Cells cultured on ground or on RPM for 24, 48 and 72 h were counted with a particle count and size analyzer (Beckman-Coulter, Inc., Fullerton, CA, USA) and by a Thoma hemocytometer. Three independent experiments in duplicate were performed. For cell cycle analysis, cell clumps were collected and centrifuged and pellets were trypsinized and washed twice with PBS (Phosphate Buffered Saline, Sigma–Aldrich, St. Louis, MO, USA). Adherent and ground control cells were trypsinized and washed twice with PBS. Cells were fixed with 70% ethanol at 4 °C for 24 h and stained with DNA PREP Stain (Beckman Coulter, Fullerton, USA) at 4 °C overnight. Stained cells were measured by flow cytometry. Cell cycle analysis was performed three times.