Penstrep antibiotic

The human breast cancer cell line MDA‐MB‐231 and murine 4T1 were purchased from ATCC. MDA‐MB‐231 and T‐47D breast cancer cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 5% fetal bovine serum (FBS, Gibco) and 1% pen–strep antibiotic (Beyotime). MCF‐7 breast cancer cells were cultured in Eagle's Minimal Essential Medium supplemented with 10%FBS and 1% pen–strep antibiotic. HEK293T cells were cultured in Dulbecco's Modified Eagle Medium (Thermo Fisher) supplemented with 10% FBS and 1% pen–strep antibiotic. All of the cell lines were tested and authenticated. All cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂. The cell lines were Mycoplasma‐free and authenticated by PCR analysis monthly.

Hydroxyl‐terminated Generation 4 (G4) and Generation 6 (G6) poly(amidoamine) (PAMAM) dendrimers were purchased from Dendritech (Midland, MI). Cy3‐ and Cy5‐mono‐NHS esters were purchased from GE Healthcare (Chicago, IL). Benzotriazol‐1‐yl‐oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), N,N‐diisopropylethylamine (DIEA), dimethylformamide (DMF), Piperidine, dimethyl sulfoxide (DMSO), trimethylamine, 6‐Fmoc‐GABA‐OH, Triton X, and bovine serum albumin were purchased from Sigma‐Aldrich (St. Louis, MO). Fischer 344 rats were purchased from Harlan Bioproducts (Indianapolis, IN), and 9L gliosarcoma cells were purchased from the Brain Tumor Research Center of UC San Francisco (San Francisco, CA). C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME), and GL261 murine glioblastoma cells were purchased from the DTP/DCTD/NCI Tumor Repository (Bethesda, MD). RPMI, fetal bovine serum, penstrep antibiotic, l‐glutamine, and normal goat serum (NGS) were purchased from ThermoFisher (Waltham, MA). Tris‐buffered saline (TBS) and phosphate buffered saline (PBS) were purchased from Corning (Corning, NY). Iba1 primary antibody was purchased from Wako Pure Chemical Corporation (Tokyo, Japan). Goat anti‐rabbit Alexafluor 488 secondary antibody was purchased from Invitrogen (Carlsbad, CA). NucBlue cell stain (DAPI) was purchased from Cell Signaling (Danvers, MA).

EFCs were generated in our lab as described previously [66 (link)]. Cells were suspended in RPMI-1640 media (Thermo Fisher Scientific, Waltham, MA, USA) and supplemented with 10% fetal bovine serum (FBS; Invitrogen, Life Technologies, Waltham, MA, USA) and 1% PenStrep antibiotic (Thermo Fisher Scientific, Waltham, MA, USA), then incubated at 37 °C in a 5% CO₂ atmosphere.
Cultured EFCs (~1 × 10⁶) were seeded and treated with GNR (A) and GNR (B) (~5.5 µg/mL, based on the viability study) and supplemented with 10% fetal bovine serum (FBS) for 48 h. Treated cells were visualized and their morphology was examined under the microscope after 48 h of incubation (Leica DMi1 inverted microscope, Leica Microsystems, Mannheim, Germany), then images of the cells were captured and compared to untreated ones.

HEK293FT cells (Thermo Scientific) were maintained in Dulbecco modified eagle media (DMEM) containing 4.5 g L⁻¹ glucose, 2 mM l-glutamine, and supplemented with 10% fetal bovine serum and 1% Pen Strep antibiotic (Thermo). Cells were maintained at 37 °C in 5% CO₂. All procedures with animals followed the guidelines approved by the Institutional Animal Care and Use Committee of National Institute on Aging. Primary neuronal cultures were prepared from newborn P0 C57Bl/6 J mice. Dissected hippocampi were incubated in 10 mL Basal medium eagle (BME) (Sigma) supplemented with 5 mL papain solution (Worthington) for 30 min at 37 °C. Brains were then incubated with 5 μg of DNAseI and titurated to dissociate single cells. Cells were washed with two cycles of 10 mL BME and counted. Cells were plated at ~0.5 × 10⁶ on Poly-d-lysine & laminine precoated coverslips in BME supplemented with B27, N2, 1 mM Glutamax (Invitrogen), 0.45% glucose (SIGMA). Media was replaced the next day with BME supplemented with 2.5 μM cytosine arabinose to kill off glial cells.

Two different human HER2-positive breast cancer cell lines (SKBR3 and ZR75-1) derived from females were obtained from American Type Culture Collection (ATCC) (Rockville, MD, USA). Cell lines were grown and expanded in RPMI-1640 (Gibco, Life Technologies) supplemented with 10% fetal bovine serum (Gibco, Life Technologies, Massachusetts, MA, USA), 2 mM l-glutamine, 1% PenStrep antibiotic (Invitrogen, Life Technologies, Carlsbad, CA, USA) at 37 °C, and 5% CO₂ humidified atmosphere. Human normal mammary epithelial cells immortalized by E6/E7 of HPV type 16 (HNME-E6/E7) were used to assess plant extract toxicity [81 (link)]. Cells were maintained in Gibco^® Keratinocyte-SFM (1X) media (Gibco, Life Technologies). All the experiments were carried out when cells were ~70–80% confluent.