Gibco glutamax

Isolated canine islets were cultured in 150 mm petri dishes in 25 mL of CMRL 1066 media (with a glucose concentration of 5.6 mM) supplemented with 10% fetal bovine serum, 2 mM glutamine (GIBCO® GlutaMAX, ThermoFisher Scientific), and an antibiotic-antimycotic (GIBCO® Anti-Anti 100X, ThermoFisher Scientific) at 37 °C and 5% CO₂ at a maximum density of 5000 islet equivalents per dish [23 (link), 24 (link)]. Media was exchanged the morning after isolation followed by every other day at a minimum of 50% by volume.

Primary mouse hippocampal and cortical cultures were prepared on embryonic day 18. The pregnant female was rapidly killed by cervical dislocation, the abdomen was sterilized, and the embryos were collected under sterile conditions. Post-decapitation the embryonic brains were quickly isolated and immersed in ice-cold Gey’s balanced salt solution supplemented with glucose and with a pH adjusted to 7.3. The hippocampi and/or cortices were dissected, incubated in Trypsin-EDTA (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 30 min and further dissociated mechanically using a Pasteur pipette. The cells were re-suspended in Gibco Neurobasal medium ( ThermoFisher Scientific, Waltham, MA, USA) supplemented with 2% B27(Invitrogen, Carlsbad, CA, USA) (v/v), 10% N2 and 0.5 mM Gibco glutamax ( ThermoFisher Scientific, Waltham, MA, USA) and plated at high (7 × 10⁴/cm²) or low density (3.5 × 10⁴/cm²) on 12 mm glass coverslips previously coated with poly-L-lysine (Sigma-Aldrich, St. Louis, MO, USA). The cultures were incubated at 37 °C, 5% CO₂ and 95% O₂ until usage. Medium change was done once a week by replacing 20% of the medium. The C57Bl/6J WT hippocampal cultures were fixed at DIV21 whereas Mecp2 and Cdkl5 cortical cultures, at DIV7.

BV2, an immortalized murine microglial cell line, was cultured in growing medium containing Dulbecco’s modified Eagle medium (DMEM) (Gibco™GlutaMAX™, Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin (Thermo Fisher Scientific) in 5% CO₂ in air at 37 °C in a humidified incubator. Cells were re-cultured every 2 days starting at a concentration of 2 × 10⁵ cells/ml in T75 flask (Sarstedt). For a large scale of EV collection, microglia were plated in T175 flask (Sarstedt). For inflammatory activation, cells were challenged with 1 μg/ml LPS (Sigma-Aldrich, Clony 0127-B8) for 12 h and then grown for 12 h in serum-free media prior to collection of EVs. For TNF inhibition experiment, microglia were plated in growing medium either with 1 μg/ml LPS, 200 ng/ml etanercept, or both for 12 h. EVs were collected from serum-free media 12 h after treatment.

The TNBC cell line 4T1 (CRL-2539) was obtained from ATCC. The D2F2/E2 cell line was a kind gift from Dr. Wei-Zen Wei from Wayne State University. D2F2/E2 cells were generated by transfecting D2F2 cells with full length human ErbB-2 (HER2) [19 (link)]. Cell lines were verified using their expression profile of surface markers and morphology. Mycoplasma testing was performed routinely as a precautionary quality check. These cell lines were cultured in complete DMEM with 10% Hyclone FBS (Cytiva, Marlborough, MA, USA), 4500 mg/L glucose (MilliporeSigma, Burlington, MA, USA), Gibco Glutamax (Thermo Fisher Scientific, Waltham, MA, USA), and Penicillin-Streptomycin (MilliporeSigma, Burlington, MA, USA). Selection medium for D2F2/E2 cells contained 800 μg/mL G418 (MilliporeSigma, Burlington, MA, USA).