96 well tissue culture treated plate

Human breast cancer cell lines MCF-7 and MDA-MB-231 were obtained from ATCC (Manassas, VA). Cancer cell lines were transfected with pVision-RFP-C Vector from BioVisions (Milpitas, CA) using Transfast from Promega (Madison, WI). Human umbilical vein endothelial cells (HUVECs) and EGM-2 medium were obtained from Lonza (Walkersville, MD). Adipose-derived, human mesenchymal stem cells (hMSC) and hMSC medium were obtained from ATCC (Manassas, VA). The basement membrane extract (BME), tumor-aligned basement membrane extract Type 3 (BME-3), 10X Spheroid Formation ECM, Tumor-aligned RPMI (TARPMI), collagen-1, and calcein am were obtained from Trevigen, Inc. (Gaithersburg, MD). RPMI 1640 was obtained from Invitrogen (Carlsbad, CA). Tissue culture flasks, 96 well ultra-low adhesion plates, and 96 well tissue culture-treated plates were obtained from Corning, Inc. (Corning, NY). Fetal bovine serum (FBS) was obtained from HyClone (Logan, UT). Fluorouracil and paclitaxel were both identified as current breast cancer treatments from the NCI Developmental Therapeutics Program [DTP] Approved Oncology Drugs Set, and these and ethidium bromide were obtained from Sigma (St. Louis, MO). The cytoplasmic membrane dyes, Neuro-DiO and DiB, were obtained from Biotium (Hayward, CA).

Viability of cells within biofilms was determined by an MTT assay (Mapes et al., 2016 (link); Grela et al., 2018 (link)) with modifications. Overnight cultures of E. coli were diluted (1:100) in tryptic soy broth (TSB) and seeded in 96-well tissue culture treated plates (Corning Inc., Corning, NY, United States). Plates were incubated at 37°C for 24 h, washed with phosphate buffered saline (PBS), and incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at a concentration of 0.45 mg/mL for 3 h in the dark. Then, the MTT was removed, and the resulting formazan crystals were dissolved with dimethyl sulfoxide (DMSO) for 10 min. The metabolic output of cells within the biofilm was determined by measuring absorbance at 540 nm in a Biotek Synergy HT (BioTek, Winooski, VT, United States). To grow biofilms in urine, overnight cultures of E. coli were diluted (1:100) in LB medium and incubated for 2 h. Cells were harvested, washed, and normalized to OD₆₀₀ = 0.03 in urine supplemented with 20 mg/mL of bovine serum albumin (BSA) (Colomer-Winter et al., 2019 (link)), seeded in 96-well plates and incubated for 24–48 h or 7 days at 37°C. Fresh BSA-supplemented urine was replenished every 24 h. Biofilm viability was determined as above.

Single-cell suspensions of splenocytes from Id1-overexpressing and control vector animals (10⁵ cells) were co-cultured in the presence of OVA_323–339 peptide with 10⁵ CD4⁺ T cells isolated from OT-II transgenic mice (C57BL/6-Tg(TcraTcrb)425Cbn/J, JAX) using the CD4⁺-negative selection kit (Miltenyi Biotec) and stained using CellTrace CFSE Cell Proliferation Kit (Invitrogen). T-cell proliferation was measured by CFSE dye dilution and cytokine production by enzyme-linked immunosorbent assay (ELISA; R&D Systems) following a 72-h incubation in 96-well tissue culture-treated plates (Corning).