L glutamine

Immortalized bone marrow-derived macrophages from B10A.Bcg^r mice (B10R macrophages) were grown and maintained at 37°C in 5% CO₂ in Dulbecco’s Modified Eagle’s Medium (Wisent Inc., St-Bruno, QC) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen, Burlington, ON, Canada), 2mM L-glutamine, 100U/ml penicillin, and 100 μl/ml streptomycin (Wisent, St-Bruno, QC, Canada). BHK-21 cells (ATCC) were grown and maintained at 37°C with 5% CO₂ in Eagle’s Minimum Essential Medium (Wisent) supplemented with 5% heat-inactivated FBS, 2mM L-glutamine, 100 U/ml penicillin, and 100 μl/ml streptomycin.
Primary antibodies purchased for immunoblotting were phospho-p44/42 (ERK 1/2) (#9101S); ERK 1/2 (#4695S); phospho-p38 (#9216S); p38 (#9212S); phospho-IRF3 (4D4G); IRF3 (D6I4C), phospho-TBK1/NAK (D52C2), and TBK (D1B4) (Cell Signaling Technology, Danvers, MA, USA).

Vero cells (ATCC) and HT1080 cells (ATCC) were cultured in Minimum Essential Medium (MEM, Sigma,) and Human Embryonic Kidney HEK293T (ATCC) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Wisent). All culture media were supplemented with 10% Fetal Bovine Serum (FBS, Sigma), 0.3 mg/mL L-glutamine, 100 U/mL penicillin, and 100μg/mL streptomycin (Wisent). Cells were maintained at 37°C in 5% CO₂ at 100% relative humidity.
BMDMs were isolated from C57Bl/6J mice (stock no. 00064, originally purchased from Jackson Laboratories and maintained as a colony at the University of Ottawa) and differentiated as previously described [73 (link)]. Briefly, bone marrow cells were seeded in DMEM supplemented with 10% FBS (Wisent), penicillin, and streptomycin (Hyclone, GE healthcare). L929-conditioned media (20%) was utilized for macrophage differentiation, and Roswell Park MEMorial Institute (RPMI, Wisent) media supplemented with 10% FBS (Sigma-Aldrich), 0.3mg/mL L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Wisent) was utilized for seeding and subsequent experiments.

In some experiments, some of the animals were sacrificed 4 weeks after the second vaccination. Spleens were collected and splenocytes were isolated as previously described with the following modifications [13 (link)]. Splenocytes were resuspended in 96-well plates (10⁶ cells/well) in RPMI-1640 (Wisent Bioproducts) supplemented with 10% fetal bovine serum, 1 mM penicillin/streptomycin, 10 mM HEPES, 1X MEM non-essential amino acids, 1 mM sodium pyruvate, 1 mM L-glutamine (all from Wisent Bioproducts), 0.05 mM 2-mercaptoethanol (Sigma-Aldrich). The cells were incubated at 37ºC in the presence of 2.5 μg/mL of rCatB for 72 hours after which the supernatant cytokine levels of IL-2, IL-4, IL-5 IL-10, IL-12p70, IL-13, IL-17, IFNγ, and TNF-α were measured by QUANSYS multiplex ELISA (9-plex) (Quansys Biosciences, Logan, UT) following the manufacturer’s recommendations.

After overnight fasting, 80mL of blood were collected in heparinized tubes by venipuncture and diluted two-fold with phosphate buffered saline (PBS). PBMCs were isolated by Ficoll–Paque™ Plus (GE Healthcare, Piscataway, NJ) density sedimentation, as described in [89,95,96]. Briefly, diluted blood was carefully layered onto the Ficoll-Paque density gradient and centrifuged for 20 min at 400 x g with slow acceleration and with the brake off at room temperature. After centrifugation, the leukocytes resolve into one band with red blood cells at the bottom of the tube. Plasma were collected, aliquoted and stored at -80°C for further assays. PBMC’s consisting of monocytes and lymphocytes were obtained from the band and re-suspended in 1x PBS. All the tubes were centrifuged at 250 x g for 5 min, and the pellets resuspended in complete medium consisting of RPMI-1640 medium supplemented with 2 mmol/L L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% foetal calf serum (Wisent Inc., St Bruno, QC). Cell viability was > 95% (Trypan blue exclusion). Identical numbers of cells from all groups were used in each comparative experiment. Cells were maintained in a complete medium for further assays.

Whole blood or leukapheresis samples were acquired from St. Michael’s Hospital, Unity Health. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation using Ficoll-Paque PLUS (GE Healthcare). PBMC were resuspended in R10 medium consisting of RPMI 1640 (Wisent), 10% heat-inactivated fetal bovine serum (FBS; Wisent), 10 mM HEPES (Wisent), 2 mM l-glutamine (Wisent), and 100 U of penicillin-streptomycin solution (Wisent). PBMC were diluted 1:1 with freezing medium consisting of 20% dimethyl sulfoxide (DMSO; Sigma-Aldrich) in FBS and aliquoted for −150°C storage. PBMC used in the T cell epitope mapping were sent to Scisco Genetics Inc. (Seattle, WA) for HLA typing (Table 3).