Peg4000

A human podoplanin cDNA region encoding amino acids 76–89 (226–267 bp) was cloned and tandemly connected 12 and 40 times. These cDNA fragments were inserted into a pGEX-6P-3 vector (GE Healthcare, Buckinghamshire, UK). Next, the GST-tagged human podoplanin peptide (76–89 aa) produced in BL21 (DE3) E. coli was purified using glutathione sepharose. Six-week-old female BALB/c mice were injected with the GST-tagged peptide as an immunogen in conjugation with Titer MAX Gold adjuvant (Titer MAX, Norcross, GA, USA). Further, intraperitoneal immunization was performed intermittently for two months. Mice were euthanized, and splenocytes were fused with mouse myeloma P3U1 cells using PEG4000 (Merck, Whitehouse Station, NJ, USA). Hybridoma screening and antibody purification from ascites were performed as described previously [22 (link)]. IgG isotypes were identified using the Mouse Monoclonal Antibody Isotyping Test Kit (AbD Serotec, Oxford, UK).

The vector pBABEhygro (Addgene) was introduced into MethA tumor cells by calcium phosphate precipitation to obtain hygromycin B resistant clones. D2SC/1 cells were similarly transfected with pBABEpuro. Transfected cells were cultured in growth medium containing 5 μg/ml puromycin or 100 μg/ml hygromycin (Life Technologies). To obtain fusion hybrid cells, 10⁷ hygromycin resistant MethA tumor cells were mixed with 5 × 10⁷ puromycin resistant D2SC/1 cells and briefly centrifuged. Cellular pellets were gently resuspended in 1 ml PEG 4000 (Merck, Darmstadt, Germany) containing 0.5 ml RPMI 1640 medium and incubated at 37°C for 90 s. Subsequently, 15 ml RPMI 1640 medium was added drop wise to the cells, then 20 ml RPMI 1640 medium with 10% fetal calf serum. After 5 min the cell suspension was centrifuged and cells were plated in RPMI 1640 medium and 10% fetal calf serum. After 24 h the fused cell hybrids were selected in the presence of 100 μg/ml hygromycin and 5 μg/ml puromycin. When colonies were developed, individual hybrid cell clones were isolated using cloning cylinders (Sigma-Aldrich) and 50 μl of trypsin-EDTA.

Silk cocoons were purchased from local market of Tabriz (Tabriz, Iran). DMSO, mannitol, sorbitol, PEG 4000, and lactose were procured from Merck (Darmstadt, Germany). 5-FU was purchased from Sigma–Aldrich (St. Louis, MO, USA). Sodium carbonate and Lithium bromide (LiBr) were supplied from Scharlau (Sentmenat, Spain) and from Ridel-de Haen (Seelze, Germany), respectively. Acetone was obtained from Dr. Mojallali (Markazi, Iran).

PEG4000 (8074850050, Merck) and cremophor (238470-1SET, Merck) were heated in a warm bath at +37 °C until fluidity increased, before both were added to NaCl 9 mg/mL in a ratio of 1:1:8. The mixture was sonicated (3 × 15 min). Trametinib (synonyms: GSK1120212, JTP-74057, Mekinist) from Selleckchem (catalog No. S2673, USA) was then added to this mixture and vortexed, creating a 10 mM solution. The mixture with Trametinib was reheated to 37 °C and sonicated until dissolved (3 × 15 min).