Rpmi cell culture media

Bioactivity of ML1 that had been encapsulated in liposomes and released by HIFU was studied in two experimental settings that reflect either the uptake of ML1 in target cells (functionality of the A-chain of ML1) or that represent the biological activity of ML1 (cytotoxin activity). All experiments were conducted with murine CT26 colon carcinoma cells that has been obtained from American Type Culture Collection (ATCC). RPMI cell culture media, PBS, and FBS were purchased from Sigma-Aldrich, and OptiMem was obtained from Gibco. CT26 cells were cultured in RPMI supplemented with 10% FBS, at 37°C in a 5% CO₂ and humidified atmosphere. For all cell experiments, CT26 colon carcinoma cells were first seeded in 96-well plates (10000 cells/well) and allowed to adhere for 24 h prior to the experiment.

Spleens were harvested from NOD female mice euthanized at 10 weeks of age. A single cells suspension was made by first physically dissociating the spleen followed by a 10 min incubation in RBC lysis buffer (eBioscience) to remove red blood cells. The cell suspension was then strained through a 100 μm filter and plated at 1 × 10⁶ cells/well in complete RPMI cell culture media (Sigma Aldrich) plus 10% fetal bovine serum (Gibco). Cells were plated in a 96-well tissue culture plate in a final volume of 200 μl and allowed to settle prior to addition of microparticles. PLGA-Red microparticles were resuspended in tissue culture media. 0.25 mg of reconstituted particles were then added to the cell wells and incubated together at 37°C for 18 h. Cells were then collected and washed three times to remove extra-cellular particles. The uptake of particles by cells was measured by flow cytometry (BD Influx) and the data were analyzed by FlowJo software version 10.7.1.