Hi fbs

MC3T3-E1 subclone 4 pre-osteoblast cells (ATCC®) passages 3–10 were maintained in growth media containing MEM-Alpha medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS, Invitrogen), 50 U/mL penicillin and 50 μg/mL streptomycin (P/S, Invitrogen). MB-MDA-231 breast cancer cells (ATCC®) passages 20–30 were maintained in high glucose DMEM (Invitrogen) supplemented with 10% HI-FBS, 50 U/mL 50 μg/mL P/S. Human mesenchymal stem cells (hMSC, Lonza) passages 2–5 were culturing using hMSC growth media (Lonza). Cells were cultured until 80–90% confluence, passaged using 0.05% Trypsin/EDTA (Invitrogen) and seeded for experiments.
To assess cell viability, 20,000 cells were incubated on the PA coatings in serum-free MEMAlpha for 4–5 hours either in a 48 well plate or on 12 mm coated glass coverslips. Cells were then exposed to calcein AM (Invitrogen) and ethidium homodimer-1 (Invitrogen) following manufacturer’s protocols. Stained cells were imaged using an inverted fluorescent microscope (Nikon, Eclipse TE2000U). For experiments with shared culture media, glass coverslips were adhered to a 60mm tissue culture plastic dish using non-toxic vacuum grease and cells were cultured for 4 hours with shared media prior to viability staining.
For LDH release, cells were seeded at 30,000 cells/well in a 48 well plate in triplicate and enzyme release was measured using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega) following the manufacturer’s protocol. Phenol-free MEM-Alpha (Invitrogen) was used to seed the cells on the coatings and the media was sampled at desired time points and normalized with a 1% Triton X-100 treated positive control, which represented 100% LDH release.
For ATP depletion experiments cells were incubated in serum free media containing 0.1% NaN₃ and 50 mM 2-deoxy-D-glucose (Sigma-Aldrich). Inhibition of myosin II activity was achieved with 50 mM blebbistatin (Calbiochem) and inhibition of actin polymerization was performed with 50 mM latrunculin A (Calbiochem). For the ATP depletion and cytoskeletal inhibition studies, adherent cells were treated in serum free media for 1 hour at 37°C. Following treatment, cells were trypsinized and added to PA coatings in serum-free MEMAlpha with the respective additives.
For cell barrier (compartmentalization) experiments, the desired number of cells were centrifuged into a pellet, resuspended in a small volume of media and further resuspended in a solution of a negatively charged PA molecule (palmitoyl-Val-Val-Ala-Ala-Glu-Glu) that was previously thermally annealed to 80°C for 30 minutes to aid with gel formation. This yielded a final PA concentration of 0.75% (w/v) and a final cell concentration that ranged from 10,000–100,000 cells/μL. The PA solution with suspended cells was then drawn into a gelling solution containing 10 mM CaCl₂, 132 mM NaCl and 3 mM KCl to make a cylindrically shaped gel, which was then broken into short pieces, immersed for 1 minute in a solution containing 0.01% (w/v) of either PA 1 or PA 2 in a solution of 147 mM NaCl and 3 mM KCl. The gels were then rinsed 3 times in PBS and were encapsulated in a 2.5 mg/mL collagen gel (rat tail, type I; BD) using a glass bottom petri dish (Mattek). Cells were cultured then fixed with 4% paraformaldehyde, permeabilized with 0.4% Triton X-100, and stained with propidium iodide (500 ng/ml, Sigma-Aldrich) to visualize nuclei and phalloidin-AlexaFluor 488 (1:100 dilution, Invitrogen) to visualize F-actin. DAPI staining (5 μg/ml, Invitrogen) aided with visualization of the PA gel, as a high degree of non-specific binding to the negatively charged PA was consistently observed.

Jurkat Clone E6–1 cells were obtained from ATCC (TIB-152). The EGFP NFAT reporter Jurkat cell line was a gift from Dr. Arthur Weiss (University of California, San Francisco). The EGFP NFκB reporter Jurkat cell line was a gift from Dr. Xin Lin (MD Anderson). The TM-LCL cell line, an Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line, was a gift from Dr. Michael Jensen (Seattle Children’s Research Institute). The OKT3⁺ TM-LCL line was a gift from Dr. Stephen Forman (City of Hope National Medical Center). The above cell lines were maintained in complete RPMI (RPMI1640 (Lonza) + 10% heat-inactivated FBS (HI-FBS, Gibco)). HEK293T cells (ATCC; CRL-11268) were cultured in DMEM (HyClone) + 10% HI-FBS. Hep G2 cells (ATCC; HB-8065) were cultured in EMEM (Lonza) + 10% HI-FBS. CD19 CAR, GFP CAR, and TGF-β CAR Jurkat cell lines were generated with lentiviral transduction followed by fluorescence-activated sorting (FACS) using the FACSAria (II) at the UCLA Flow Cytometry Core Facility. Lentivirus was produced using HEK293T cells as previously described^{50 (link)}.