Cd 1 nude mice

Heterozygous female CD-1 nude mice (Charles River, strain code 087) were intercrossed to homozygous male CD-1 nude mice (Charles River, strain code 086) to generate homozygous, athymic, nude males for use in xenograft experiments. The potential for B4 (passage 42) and C10 (passage 38) cells to form xenograft tumors was tested. Each cell line was tested in three different mice; mice that received B4 cells were littermates 47 days in age, and mice that received C10 were littermates 54 days in age. Mice were anesthetized with inhalant isoflurane for the injections. Each mouse received 5 million cells subcutaneously in the right flank. Cells were diluted in 200 μL of a 1:1 ratio of PBS and matrigel basement membrane mix (Corning). For control, 200 μl of 1:1 ratio of PBS and matrigel without cells was injected subcutaneously in the left flank. Tumor development was monitored for 6 months and diameter measurements were obtained weekly with manual calipers. Mice were euthanized via C0₂ asphyxiation (per IACUC approved protocol), necropsies were performed, and subcutaneous tumors were harvested and fixed in 4% paraformaldehyde. Paraffin-embedded sections were stained with hematoxylin and eosin and analyzed by a board-certified veterinary pathologist (TS).

In order to validate the ability of our cell lines to form sarcomas in vivo, subcutaneous xenografts in mice were evaluated using methods previously described (Borlle et al., BMC Veterinary Research 2018, in review). Female, heterozygous CD-1 nude mice (Charles River, Strain code 087) were crossed with male, homozygous CD-1 nude mice (Charles River, Strain code 086). Resulting, male, athymic, nude littermates were injected subcutaneously in the right flank with 5 × 10⁶ FISS cells of a given cell line suspended in 200 μl of a 1:1 solution of PBS and Matrigel (Corning) for each cell line. As a control, 200 μl of a 1:1 solution of PBS and Matrigel (Corning) without any cells was injected subcutaneously in the left flank. Tumor growth was monitored for up to 24 weeks. At the end of the monitoring period, mice were euthanized via carbon dioxide asphyxiation and necropsies were performed to collect tumor tissue. Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin for histopathologic evaluation.