Bovine serum albumin (bsa)

All culture media including M199, α-MEM, M5, Trypsin (0.25%)-EDTA (0.02%) and PBS, as well as Penicillin (10,000 U/ml)-Streptomycin (10,000 μg/ml) concentrate solution were prepared and supplied by the Memorial Sloan-Kettering Cancer Centre Culture Media Core Facility (New York, NY). Amphoteracin B was purchased from Life Technologies (Grand Island, NY). Gelatin was from TJ Baker Inc (Philipsburgh, NJ). Bovine serum albumin was from Gemini Bioproducts (West Sacramento, CA). Falcon tissue culture flasks and centrifuge tubes were purchased from BDBiosciences (Two Oak Park, Bedford, MA). HDF were from The Coriell Institute (Camden, NJ). SAOS-2 osteosarcoma cells were from the American Type Culture Collection (VA, USA). A673 osteosarcoma and H3122 lung carcinoma cells were from the collection at the Memorial Sloan Kettering Cancer Center. The lipophilic fluorescent probes DiD (excitation 644nm, emission 665nm) and DiO (excitation 484nm, emission 501nm) Vybrant cell labelling solutions were from Molecular Probes, Life Technologies (Grand Island, NY). DAPI was provided by the FACS core facility at Memorial Sloan Kettering Cancer Center.

Equal amounts of protein (10–20 μg) were separated through 10% Bis-Tris gel (Invitrogen, Carlsbad, CA, USA) and transferred to nitrocellulose membranes. Membranes were blocked for 1 h in 5% (w/v) suspension of bovine serum albumin (BSA; Gemini Bio-Products, West Sacramento, CA, USA) in 0.2% Tween 20-Tris-buffered saline (TBS) (pH 7.5). After blocking, the membranes were incubated overnight at 4 °C, with one of the following primary antibodies: 6E10 (1:1000; BioLegend, San Diego, CA, USA; Catalog # 83001, Lot #B2261151), anti-APP-CT20 (1:1000; EMD Millipore, Burlington, MA, USA; Catalog #171610, Lot #D00080225), anti-APP-22C11 (1:1000; EMD Millipore, Burlington, MA, USA; Catalog #MAB348, Lot #2280425. Membranes were washed in 0.2% Tween 20-TBS for 20 min and incubated at 20 °C with the specific secondary antibody anti-rabbit IgG (H + L), HRP conjugate (1:10000 Invitrogen, Carlsbad, CA, USA; Catalog #31460, Lot #RL240411) and anti-mouse IgG (H + L), HRP conjugate (1:10000 Invitrogen, Carlsbad, CA, USA; Catalog #31430, Lot#RJ240410) for 60 min. Blots were developed using Super Signal (ThermoFisher Scientific, Rockford, IL, USA) and signal value were measured with ImageJ (64-bit) software.

For MMGO handling, plasticware was pre-coated with PBS supplemented with 2.5 mg mL⁻¹ bovine serum albumin (BSA, #700-100P from Gemini Bio-Products). Mammary glands were collected, and lymph nodes removed. Mammary glands were then minced with scissors and digested in DMEM/F12 medium supplemented with 5% FBS, 50 µg mL⁻¹ gentamicin sulfate, 5 ng mL⁻¹ insulin, 0.04% (w/v) Trypsin-EDTA (#15400054, Thermo Fisher) and 2 mg mL⁻¹ collagenase A (#10103586001, Roche) for 1 h at 37 °C at 100 rpm in a HulaMixer sample mixer (Life Technologies). Thereafter, mammary glands were subjected to vigorous shaking to disrupt adipocytes, and cells were pelleted at 520 g for 10 min. After further washing in DMEM/F12 medium, both the cell pellet and the fat layer were recovered. The latter was mechanically dissociated, pelleted again, and then added to the cell pellet. Thereafter, cells were resuspended for 5 min in DMEM/F12 medium supplemented with 4 U mL⁻¹ DNase I (#DPRF, Worthington Biochemical), pelleted, and cleared by differential centrifugation. Mammary organoids were finally collected and embedded in Matrigel® Matrix (#356255, Corning) for 3D culture.

Immortalized human bronchial epithelial cells (BEAS-2B) were purchased from ATCC (Manassas, VA) and maintained in Bronchial Epithelial Cell Medium (BEBM) with growth supplements (Lonza, Walkersville, MD). BEAS-2B cells require special coating on the flasks with a mixture of 0.01 mg/mL fibronectin (Sigma, St. Louis, MO), 0.03 mg/mL bovine collagen type I (Advanced Biomatrix, San Diego, CA) and 0.01 mg/mL bovine serum albumin (Gemini Bio-Products, West Sacramento, CA) dissolved in BEBM. NCI-H460 cell line was purchased from ATCC and maintained in RPMI-1640 with 10% Fetal Bovine Serum (Gemini Bio-Products, West Sacramento, CA), 1% streptomycin-penicillin (Invitrogen, Carlsbad, CA). Human monocytic cell line (THP-1) was purchased from ATCC (Manassas, VA) and maintained in RPMI-1640 supplemented with heat inactivated 10% FBS (Gemini Bio-Products, West Sacramento, CA). All the cell types were maintained and treated at 37°C in a humidified incubator in presence of 5% CO₂. Each cell line was maintained in culture for only 4–5 passage numbers for a given experiment. Short Tandem Repeat (STR) DNA profiling was used to confirm the authenticity of each cell line.

Borrelia isolates were cultivated in BSK media (33°C; 5% CO₂) prepared using bovine serum albumin from Gemini Bio-Products, Inc. (lot #C54) as previously described (Samuels et al., 1994 (link)). All genetically manipulated strains were initially cultivated in BSK media or BSK semi-solid media plates containing 75 μg/mL streptomycin or 75 μg/mL kanamycin, as appropriate. BL21 (DE3) and TOP10 E. coli cells were cultivated in LB Broth, Lennox, or LB agar (Fisher BioReagents) containing 100 μg/mL ampicillin at temperatures detailed below.