Trypsin edta

RPMI-1640 media containing mycoplasmas (1x10⁵ CFU/ml) were pre-incubated with boiled CA27 (3 μg/ml) and increasing concentrations (1–3 μg/ml) of CA27 at 37°C for 3 hours, added to mycoplasma-free Huh7 cells (2 x 10⁵ cells/well) in 12 well plates, and further incubated at 37°C for 2 days in a 5% CO₂ incubator. After washing three times with phosphate buffered saline (PBS, pH7.4), cells were detached by 0.25% Trypsin-EDTA (Welgene). The cells (1×10⁴ cells) were washed with PBA (PBS plus 0.1% bovine serum albumin) and analyzed by flow-cytometric analysis with CA27 followed by a further incubation with fluorescein isothiocyanate (FITC)-conjugated mouse IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at room temperature (RT). CA27 binding to mycoplasma-infected cells was determined by flow cytometry and shown as mean fluorescence intensity (MFI). To detect mycoplasmas by polymerase chain reaction (PCR) technique, infected Huh7 cells (2 x 10⁵ cells) were suspended in distilled water after harvest. Suspended cells were boiled at 100°C, and the supernatants were then subjected to PCR using e-Myco™ VALid-Q mycoplasma qPCR detection kit (Intron, Seoul, Korea). The amplified PCR products were visualized by agarose gel electrophoresis analysis.

Polydatin was purchased from J&H Chemo Co., Hangzhou, China. RPMI-1640 medium, phosphate-buffered saline (PBS), fetal bovine serum (FBS), and trypsin-EDTA were purchased from Welgene Inc., Korea. Antibodies against NLRP3 (# 13158), phospho-p65 (# 3031), p65 (# 8242), Lamin A (# 4777), α-tubulin (# 2125), COX-2 (# 4842), caspase-1 (# 3866), SOD1 (# 4266), GPx (# 3206), HO-1 (# 43966), and β-actin (# 3700) were purchased from Cell Signaling Technology (Danvers, MA, USA). The ROS inhibitors N-acetyl-l-cysteine (NAC) and H₂DCFDA were purchased from Sigma Chemical Co., St. Louis, MO, USA.

For analysis of cell viability, MTT assay was carried out by a procedure similar to that described previously [30] (link). Briefly, cells were seeded in 24-well plate (5×10⁴ cells/well) and grown for 24 h. The cells were then treated with MML at different concentrations (0-80 µM) for 24 h or treated with 60 µM of MML for different time courses (0-24 h). After MML treatment, cells were incubated with medium/MTT (0.5 mg/ml) mixture for 3 h. DMSO was added to dissolve the reduced formazan crystal from MTT and absorbance was read at 540 nm using ELISA plate reader (Bio-Rad, Hercules, CA, USA). To confirm the effect of MML on cell proliferation, live cells were counted by trypan blue staining according to the manufacturer's instructions. In brief, A549 cells were plated in 12-well plate and treated with MML as indicated concentrations for 24 h. After treatment, cells were harvested using trypsin-EDTA (WelGENE Co., Daegu, Korea) and stained with 0.4% trypan blue solution (Gibco, Carlsbad, CA). Living cells were counted by hemocytometer. Cell viability was shown relative to the control.

To assess the proliferation ability, a cell proliferation assay was performed using cell-counting and WST-1 assays. After transfection, the cells were seeded in six-well plates to perform the cell-counting assay. After 2–3 day, cells were washed with PBS. 0.25%. Trypsin EDTA (WelGENE, Korea) was added for 2 min at 37 °C and then neutralized with complete growth media. The cells were diluted in trypan blue and counted daily. To evaluate the effect of SEMA3C on the proliferation assay, cells were seeded into serum-free media in six-well plates. After 24 h, the SEMA3C (50 ng/mL) was treated for 24 h, and the cells were counted with trypan blue staining. Treatment with 10% FBS was used as a positive control.
For the WST-1 assay, transfected cells were seeded at a density of 1 × 10⁴ cells/well into 96-well plates for 24–72 h. Ten microliters of EZ-Cytox (Deaillab, Seoul, Korea), a WST-1 reagent, was added into each well per day. After incubation for 30 min at 37 °C, the absorbance was measured at 450 nm.

Human embryonic kidney 293T (HEK293T) cells were bought from the American Type Culture Collection (ATCC) and grown in Dulbecco’s modified Eagle’s medium (DMEM) (Welgene, Republic of Korea) with 10% fetal bovine serum (FBS) (Welgene) and 1% penicillin-streptomycin (Gibco, USA). The cells were maintained at 37°C, in a 5% CO₂ humidifying incubator. Dulbecco’s phosphate-buffered saline (DPBS) and 0.05% Trypsin-EDTA purchased from Welgene were also used.

B16-F10 mouse melanoma cells were cultured in Dulbecco's Modified Eagle Medium (Welgene) containing 10% fetal bovine serum (Welgene) and 1% penicillin/streptomycin (Welgene). Once cells reached approximately 80–90% confluency, cells were harvested using Trypsin/EDTA (Welgene) and rinsed with PBS (GenDEPOT). The flanks of mice were subcutaneously injected with 5 × 10⁵ cells in 100 μl PBS. Ten days after tumor inoculation, mice were randomized into four groups that were injected with 20 μl of PBS (n = 6), anti-CD25-Ce6 (n = 6), isotype-Ce6 (n = 5), or free anti-CD25 (n = 6) intratumorally four times total at 2-day intervals. The tumors were injected with anti-CD25-Ce6 or isotype-Ce6 and irradiated (660 nm, approximately 100 mW/cm², 30 min) by laser 30 min after injection. Tumor volumes were measured at 3-day intervals for 24 days by a blinded investigator with calipers using the following equation: Tumor volume = (L × W²)/2, where L indicates the length of the long side and W indicates that of the short side.