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For the immunization of F2-hybrid mice, 0.5 mg of C3a peptide in 1 mL of PBS was mixed with equal volume of Freund’s complete adjuvant (or Freund’s incomplete adjuvant for reimmunization). The reimmunization was carried out after 30 days; 12 days after that, the lymphocytes from the inguinal, peritoneal and axillary lymph nodes were collected for hybridization with Sp2/0 myeloma cells in the ratio 2:1. Cell hybridization was performed in PEG/DMSO (Sigma), using serial dilution with culture media [31 (link)]. The hybrids were transferred to 96-well culture plates, and cultivated for 12–16 days in RPMI-1640 medium (Sigma) containing 20% fetal calf serum. The HAT supplement (Sigma) was added to the culture medium solution for selection of hybridomas.
Clone screening was performed using ELISA in a 96-well microplate. Briefly, 50 µL of culture media was added to wells precoated with C3a antigen, and after incubation for 1 h, peroxidase-conjugated goat anti-mouse IgGs were added. Detection was performed with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution.
For C3a ELISA development, two monoclonal murine antibodies to C3a, with different epitope specificities designated as CC3a-5 and CC3a-1, were selected. CC3a-5 was immobilized on a solid phase as a capture antibody, while CC3a-1 was conjugated with horseradish peroxidase and was used as a detection antibody.

10⁷ BCBL-1 cell aliquots were diluted in 200 μL sterile PBS, and 6-8 week-old male non-obese diabetic/severe-combined immunodeficiency (NOD/SCID) mice (Jackson Laboratory) received intraperitoneal (i.p.) injections with a single aliquot. For drug delivery, dhC16-Cer or C6-Cer was diluted in sterile PEG:DMSO (1:1) (Sigma) to achieve 100 μL total volume. The drug, or vehicle alone, was administered using an insulin syringe for i.p. injections. Drug was administered initially at either 1 day or 28 days after BCBL-1 injections, 3 times/week. Two experiments, with 10 mice per group for each experiment, were performed. Weights were recorded weekly as a surrogate measure of tumor progression, and ascites fluid volumes were tabulated for individual mice at the completion of each experiment. All protocols were approved by the Louisiana State University Health Science Center Animal Care and Use Committee in accordance with national guidelines.