Penicillin streptomycin glutamine psg

The normal mammary epithelial cell line MCF-10A (American Type Culture Collection, ATCC CRL-10317, Manassas, VA, USA) and the triple-negative breast cancer cell line MDA-MB-231 (ATCC HTB-26) were cultured using standard tissue culture conditions as previously reported (Gaur 2021). An additional triple-negative breast cancer cell line, HCC1806 (ATCC CRL-2335), was cultured and maintained in RPMI-1640 media (Sigma Aldrich, Louis, MO, USA) with 10% fetal bovine serum (FBS, Gemini Bio, West Sacramento, CA, USA) supplemented with 1% Penicillin–Streptomycin–Glutamine (PSG, ThermoFisher Scientific, Louis, MO, USA). Before the initiation of infection, cells were synchronized to the G0 phase of the cell cycle by incubating in serum-free media for 10 hours. The cells at G0 phase represent a quiescent phase in which cells are not actively dividing, thus allowing analysis of the initial events of the cell cycle [27 (link)]. In addition, synchronizing cells reduces the variability between cells, creating a more precise way of comparing experimental results and achieving increased reproducibility of experiments [27 (link)]. Following synchronization, a total of one million cells were infected with B. burgdorferi with a multiplicity of infection (MOI) of 60 as previously described [20 (link)].

HEK293 cells were seeded onto 24-well plates (1.0 × 10⁵ cells per well) in Dulbecco’s modified Eagle’s medium (DMEM) containing Penicillin-Streptomycin-Glutamine (PSG, Thermo Fisher Scientific) supplemented with 10% (v/v) fetal calf serum (FCS) in 5% CO₂ at 37 °C and cultured to grow to 80% confluence for 24 h. After 24 h, cells were rinsed with PBS and conditioned medium was replaced with PSG-free DMEM supplemented with 10% (v/v) FCS.
HEK293 cells were transiently transfected with pCAGGS (an empty vector plasmid) or pCAGGS containing CYP cDNA (400 ng per well) and co-transfected with CYPOR (100 ng per well) using ViaFect Transfection Reagent (Promega). Transfected cells were cultured for 24 h under the same conditions as described. After 24 h, transfected cells were rinsed with Hanks’ balanced salt solution (HBSS, with Ca²⁺ and Mg²⁺, Thermo Fisher Scientific) supplemented with 0.1% (w/v) bovine serum albumin (BSA, Sigma-Aldrich), which was essentially fatty acid free, and treated with EPA, DHA, and AA (30 μM each) in HBSS. Treated cells were incubated at 37 °C for 1 h, and after incubation ice-cold methanol was added to stop the reaction. Culture supernatant and methanol were collected as samples.

Cell lines were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) and were authenticated by their short tandem repeat profile. All cell lines were tested monthly for Mycoplasma contamination with the MycoAlert Mycoplasma Detection Kit (Lonza, LT07-705) according to the manufacturer's instructions. Cells were passaged twice a week and cultured in RPMI 1640 medium (PAN Biotech, P04-16500) media supplemented with 10% fetal calf serum (Thermo Fisher Scientific, 10270106), 1% HEPES (Carl Roth, HN78.1) and 1% penicillin-streptomycin-glutamine (PSG) (Thermo Fisher Scientific, 10378016) at 37°C in a 5% CO₂ atmosphere. Cells were used within 2 months of thawing.

PBMCs were purified from whole blood samples using Ficoll (GE Healthcare) gradient centrifugation and cryopreserved in 90% Fetal Bovine Serum (FBS; HyClone) containing 10% dimethyl sulfoxide (DMSO; Fisher). Frozen PBMCs were thawed and cultured in Roswell Park Memorial Institute (RPMI)-1640 complete media (GenClone) supplemented with 20% heat inactivated FBS, 1x penicillin-streptomycin-glutamine (PSG; Thermo Fisher Scientific), and 5% (5 U/mL) human rIL-2 (NIH AIDS Reagent Program). Cells were induced with 5 μg/mL phytohaemagglutinin-P (PHA-P) (Sigma) for 48 h at 37°C and 5% CO₂.