Gentamycin

The D10 strain of P. falciparum was cultured in O⁺ human RBCs (Interstate Blood Bank, TN) supplemented with RPMI 1640 medium along with 0.3–0.5% Albumax I (Invitrogen), 2g/L sodium bicarbonate, 10 mg/L hypoxanthine (Thermo Fisher Scientific), 15 mM HEPES (Millipore Sigma) and 50 mg/L gentamycin (VWR). Parasite culture was maintained at 37°C with conditions previously described [17 (link)].

Bone Marrow derived Dendritic Cells (BMDCs) infected with Listeria were used as antigen presenting cells in the T cell activation assay. BMDC were generated as previously described (53 (link)). Briefly, bone marrow was harvested from 8–12-week old C57BL/6 mice and cultured in the presence of 10 µg/ml recombinant Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) BMDCs were infected with Lm-10403s at a MOI of 1, and after 1 h, 20 μg/ml gentamicin (VWR) was added to the culture to inhibit bacterial replication (this concentration of gentamycin inhibits both extracellular and intracellular bacterial growth in our system, data not shown). The cells were incubated for 24 h at 37°C with 5% CO₂ to allow antigen processing and presentation before co-culture with T cells. Splenocytes from infected or uninfected mice were cocultured with DCs at a ratio of 10:1 for 5 h in the presence of GolgiStop (monensin) (BD Biosciences). Cells were washed in FACS buffer (PBS supplemented with 3% FBS) and were incubated with AF488-anti-CD3 at 4°C for 10 min. The cells were washed in FACS buffer twice then fixed and permeabilized (BD CytoFix/CytoPerm) by incubating for 20 min at 4°C. The cells were washed in Perm/Wash buffer and incubated with PE-anti-IFN-γ for 30 min at 4°C. Cells were washed twice in Perm/Wash buffer and resuspended in FACS buffer before flow cytometric analysis.

The protocol was adapted from Reference [22 (link)]. In a 96-well flat-bottomed plate, 100 μL of twice the selected antibiotic concentration was added to 100 μL of MRS broth with (0.1%) l-cysteine and used to perform two-fold serial dilutions. The wells were then seeded with 100 μL of 10⁵ CFU/mL of the selected bioactive strains. The absorbance at 600 nm was measured using Tecan Microplate Reader Spark^® (Grödig, Austria) at 0 h and then incubated in the anaerobic chamber for 24 h. Then absorbance was read once again. The minimum inhibitory concentration (MIC) was determined as the lowest concentration of the antibiotic that inhibited the visible growth of the microorganism. The antibiotics tested were ampicillin, vancomycin, gentamycin, streptomycin, erythromycin, tetracycline, and chloramphenicol (All antibiotics were obtained from Alfa Aesar, Mississauga, Canada apart from gentamycin, which was from VWR, New York, NY, USA).

HEK-293T cells were cultured in Dulbecco’s modified Eagle’s medium (Lonza, Walkersville, MD), supplemented with 10% fetal bovine serum (FBS; Corning, Manassas, VA) and 10 µg/mL gentamycin (VWR, Radnor, PA). Most suspension cells (APK-1, BC-2, BC-3, BC-5, VG-1, CRO/AP5, BCLM, HBL-6, KMS-12-BM, and MEG-01) were maintained in RPMI-1640 (Lonza) containing 20% FBS or Serum Plus-II (Sigma), 10 µg/mL gentamycin, and 0.05 mM β-mercaptoethanol (Bio-Rad, Hercules, CA). The following cell lines were grown in similar medium but were supplemented with 10% FBS or Serum Plus Medium: BJAB, BC-1, JSC-1, BCBL-1, Raji, and Daudi. All suspension cell lines were maintained at concentrations between 2 × 10⁵ and 12 × 10⁵ cells/mL during routine culture and experiments, and were routinely tested for potential mycoplasma contamination. BC-1, BC-2, BC-3, BCBL-1, KMS-12-BM, and BJAB were validated by short tandem repeat (STR) profiling through the Northwestern University NUSeq core facility. For other PEL cell lines reference STR profiles are not available and these cell lines were therefore validated by PCR for KSHV or EBV infection status.