At day 18, all tumors had reached a size ≥ 10 mm, but no tumor was >30 mm, and animals were randomized into different treatment groups. The inoculation was performed during general anesthesia with isoflurane inhalation.
The estimated mean weight of the animals at the time of inoculation was 120 to 150 g, depending on age. Rats were allowed to acclimatize for 1 week, after which tumor cells (50,000 NS1 cells) were subcutaneously inoculated in the hindleg at experimental day 0 with the animals in prone position. Seven- to 10-week-old female Fischer 344 rats (Fisher Scientific, Schwerte, Germany) were purchased and housed in pairs in cages (Taconic type 3 cages, 2 animals/cage) with access to water and fed ad libitum with rat chow. F, Dose profiles at 4-mm depth and percentage depth dose curves for CONV-RT (red) and FLASH-RT (blue) measured in a polystyrene phantom placed inside a PMMA box to mimic the rat irradiation setup. Rats were placed in PMMA boxes and positioned 1-by-1 in close connection to the Cerrobend plate. E, For irradiation, an electron applicator with source-to-applicator-end distance of 65 cm was attached to the gantry head of a clinical linear accelerator and fitted with a Cerrobend plate for beam collimation. D, Treatment parameters for CONV-RT and FLASH-RT. Animals were inoculated in prone position on day 0 and irradiated with either CONV-RT or FLASH-RT on day 21, 24, and 28. The box-and-whiskers plot presents the resulting distribution of tumor size across groups. B, Animals were randomly assigned between groups (n = 9-10) according to tumor size on day 18 after inoculation, and a Kruskal-Wallis analysis did not show any statistical difference between the groups. Tumor cells are marked with Hoescht nuclear staining (right panel), and the GFP autofluorescence can be detected (middle panel). A, The rat glioma cell line NS1 is a green fluorescent protein (GFP)–positive tumor cell line (left panel). 1 Study design to compare hypofractionated FLASH radiation therapy (FLASH-RT) and conventional RT (CONV-RT) in a subcutaneous rat glioma model. The cells were centrifuged, the supernatant was removed, and the cell pellet was resuspended to achieve the concentration used for inoculation, 1000 cells/µL.įig. Thereafter, medium was added, and viable cells were counted. Trypsin (Invitrogen) was added, and cells were incubated at 37☌ for 1 to 2 minutes to detach the adherent cells from the flask. After culturing in T25 flasks, the cells were prepared for inoculation by removal of the medium and washed gently with phosphate-buffered saline. In preparation for inoculations, cells were cultured using Iscove's Modified Dulbecco's Medium with addition of 1% mL Na-pyruvate, 1% mL HEPES (4-1-piperazineethanesulfonic acid), 0.1% mL gentamycin, and 10% inactivated fetal calf serum (heated to 56☌ for 30 minutes). Cells were mounted with Eukitt Quick-hardening mounting medium (Sigma-Aldrich), stained with Hoechst (Thermo Scientific) and photographed with a fluorescent microscope fitted with the appropriate wavelength filters ( Fig. The medium was removed, and the cells were fixed in 4% paraformaldehyde. To verify the GFP signal, cells were cultured for 1 to 2 days in 2-chamber culture slides (Thermo Fisher Scientific) at 37☌ in a humidified incubator with 5% CO 2. Sandwich enzyme-linked immunosorbent assay was used to rule out mycoplasma infection in cells and supernatant and was used according to the manufacturer's instructions (MycoProbe R&D Systems).
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