27 gauge needle

To assess malignant transformation, UROtsa cells chronically exposed to 50 nM MMA(III) from 4 to 20 weeks were injected subcutaneously (1 × 10⁷ cells per condition) into the lower right flank of the mouse in a total volume of 100 μl of sterile saline using a 27-gauge needle (Becton Dickinson, Franklin Lakes, NJ). Four mice were used per condition. At 60 days from the time of injection, tumor volumes were measured. The tumor volumes (mm³) were estimated in accordance with the ellipsoid volume formula, 4/3 × π × a² × b, where a is the length of minor axis and b is the length of the major axis [68] (link). Mice were euthanized by carbon dioxide inhalation until listless and without respiration, followed by cervical dislocation or exsanguination. Tumors were harvested from each animal and portions of the tumors were placed in 10% neutral buffered formalin for 24 h then transferred to 70% ethanol. The tissues were taken to Cellular Imaging Facility Core at the University Health Sciences Center. To evaluate histology, tumor samples were paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E), and analyzed via light microscopy by Dr. David G. Besselsen (Veterinary Pathologist, University Animal Care, University of Arizona). All procedures were performed in accordance with approved protocols of the University of Arizona Institutional Animal Care and Use Committee.

PANC-1-Luc tumor cells were resuspended at 1 × 10⁴ cells/50 μl of sterile PBS. The pancreas was expressed though a 1-cm incision made in the skin and musculature of NSG mice anesthetized with continuous isoflurane (2%). Tumor cells (50 μl) were injected slowly into the head of the pancreas over the course of 30 s using a 27-gauge needle (Becton Dickinson). The needle was left for 30 s and then slowly removed using a twirling motion. The musculature was closed using with 5-0 dissolvable suture (Stoelting) in a simple uninterrupted pattern, and the skin was closed with autoclips. Meloxicam was administered preemptively and for 3 days after tumor implantation. Seven days later, tumor engraftment was confirmed using IVIS imaging. Mice were then allocated into experimental groups normalized for baseline tumor burden. CAR-T cells (10 × 10⁶) were then injected intravenously into the lateral tail vein, followed by another injection 7 days later. Tumor burden was tracked via IVIS imaging for 100 days. Survival was simultaneously tracked as well.

Bone marrow(BM)-derived DCs were obtained from WT BALB/c (H2-d) mice. Femurs and tibias from 7–to-10-week-old female mice were removed and BM cells were flushed out with PBS using a 27-gauge needle (Becton Dickinson, Cat# 302200). Red blood cells were lysed from the cell suspension with lysis buffer (Sigma-Aldrich, Cat# R7757). BM cells (5x10⁶) were seeded per well in a 6 well plate (Helena bioscience, Cat# 92006) in DC medium as described below.