Uplfln 4 objective

Cell spheroids were formed in round-bottomed 96-well tissue culture plates. Briefly, a mixture of 1 × 10⁴ cells, 80–90% unlabeled cells and 10–20% hypoxia fate-mapping cells derived from orthotopic tumors, were plated per well in spheroid formation media 1:1 DMEM (Sigma-Aldrich) and Methocult H4100 (STEMCELL Technologies). Plates were centrifuged at 1200 rpm for 7 min, twice. After 72 hr of incubation, each spheroid was transferred to a Petri dish, where they were individually isolated with the collagen solution (2 mg/mL) and quickly transferred to the center of a semi-cross-linked collagen gel in a 96-well plate at 37°C. After complete cross-linking, warm media was added. Following 4 days in culture, spheroids were imaged in an environmentally controlled microscope every 15 min for 16 hr using an Olympus (UPLFLN 4×) objective in Cytation 5 (BioTek Instruments). Cell trajectories were tracked using MetaMorph software to obtain x,y coordinates at each time. More details are available in the studies by Ju et al. (2017) (link) and Valencia et al. (2015) (link).

Female 5- to 7-week-old NOD-SCID Gamma (NSG) mice were used according to protocols approved by the Johns Hopkins University Animal Care and Use Committee. Mice were anesthetized by the intraperitoneal (i.p.) injection of 100 mg/kg Ketamine, 16 mg/kg Xylazine, Vet One. MDA-MB-231 hypoxia fate mapping cells (2 × 10⁶⁾ were injected into the mammary fat pad (MFP) closest to the second nipple. Mice were i.p. injected with 1.25 mg of pimonidazole in saline (12.5 mg/mL) (Hypoxyprobe-1) 1 hr prior to sacrificing. Tumors were excised at various time points, formalin fixed (Sigma-Aldrich) for 1 hr, saturated in 30% sucrose (Sigma-Aldrich) at 4°C overnight, embedded in OCT media (Fisher Scientific), frozen in liquid nitrogen, sectioned via a cryotome CM1100 (Leica), and mounted onto Superfrost Plus microscope slides (Fisher Scientific). Tumor tissue sections were stained with DAPI (4′,6-diamidino-2-phenylindole) (1:1000 for 15 min, RT) and mounted with anti-fade solution. To assess the entire cross section of the tumor, slides were imaged with an Olympus (UPLFLN 4×) objective using Cytation 5 microscope (BioTek Instruments). Multiple image tiles were linearly stitched with Gen5 Software (BioTek Instruments).