Pimonidazole hcl

The 3LL-based multicellular spheroids were prepared as described elsewhere with some modifications.^{47 (link)} Briefly, 50 μl of DMEM (Gibco) containing 1% (w/v) agarose was added to, and solidified in, each well of a 96-well microplate in which 3LL cells were seeded at 1 × 10⁴ cell/well and cultured for 4 – 5 days to obtain 3D multicellular tumor spheroids with 400 – 500 μm in diameters. The spheroids were carefully transferred to glass bottom dishes where Cy5-labeled nanocages (40 nM) were added and incubated for 2 h. The competition assay was performed by incubating the spheroids with the Cy5-labeled nanocages, either non-PEGylated or PEGylated FTn, in the absence or presence of 15-fold molar excess of unlabeled FTn for 1 h. After washing with PBS, Z-stack images of the spheroids were acquired at 10 μm intervals using a confocal microscope (Carl Zeiss) and subsequently 3D-rendered images were constructed using a ZEN microscopy software (Carl Zeiss). All image-based quantitative analyses were performed using ImageJ (NIH). For the microscopic observation of hypoxia, pimonidazole HCl (200 μM; Hypoxyprobe) was additionally added to the spheroids for a 2 h incubation and cryosectioned to be immunostained using the Hypoxyprobe-1 kit (Hypoxyprobe) following the manufacturer’s instruction.

Formulated microgels were initially resuspended at a density of 50 mg mL⁻¹ in fibroblast growth media (Dulbecco’s modified eagle medium (DMEM) + 10% fetal bovine serum (FBS)) and allowed to swell for 24 h at 37°C. The total number of microgels was then determined via hemocytometer counting. Aggregates were next prepared by seeding cells with increasing number of chitosan-PFC microgels to achieve incorporation ratios of 400:1, 150:1 and 50:1 (cells:microgels).Controls included spheroids with no microgels incorporated (none) and spheroids with chitosan microgels incorporated at the highest ratio (50:1). Aggregates were formed by spinning 96 U-bottom well plates in a centrifuge at 250 G for 5 min at RT. The 3D cell structures were grown for 4 days in an incubator at 37°C and 5% CO₂. On the 4th day, to visualize regions of hypoxia within the spheroids, half of the samples were incubated with 100 μM pimonidazole HCl (Hypoxyprobe Inc., Burlington, MA) in media at RT for 1-2 hr. Spheroids were then fixed using 3.7% paraformaldehyde. After fixing spheroids were harvested and frozen in OCT (Tissue-Tek, Sakura Finetek USA, Torrance, CA), sectioned in a cryostat at 5 μm, stained (details below), and then imaged using an epifluorescent microscope.