Hanks balanced salt solution (hbss)

Fresh testicular tissues recovered from castrated animals were transported to the lab at 4 °C in HBSS (Sigma-Aldrich, H9269) containing 10 units/mL of penicillin and 10 μg/mL of streptomycin (Thermofisher Scientific, Ghent, Belgium, 15140122) and dissected in small fragments. Tissues from four animals were digested in HBSS supplemented with 1 mg/mL of collagenase D (Sigma-Aldrich, 11088858001) and 1 mg/mL of DNase I (Sigma-Aldrich, 11284932001) for 20 min at 37 °C with repeated flushing every 3–4 min. Cellular suspensions were passed through a 70-μm cell strainer (VWR, 732-2758) and rinsed two times with HBSS to eliminate residual collagenase and DNase. Following counting with trypan blue and a Bürker chamber, testicular cells were used for preparation of TOs.

Cells were washed and incubated at 37 °C with HBSS (ThermoFisher, 14025-050). Subsequently, 1 µCi 3H-inosine (Hartmann Analytic, ART0738) per well was added and the cells were incubated for 5 min. ³H of the supernatant was counted using a Beckman Counter. The cells were washed with PBS, lysed with Triton X dilution (VWR, 28817.295; 1:1000 in HBSS) and ³H of the lysate was counted. Data were normalized to the protein concentrations of respective wells.

GBM cell lines were cultured in medium containing 50nM, 100nM, or 200nM UNC2025, or DMSO (vehicle control) for 120 hours, then harvested with 0.25% trypsin and 0.1% EDTA in HBSS (cat # MT-25-053-Cl) purchased from VWR (Radnor, Pennsylvania) and resuspended in 500uL of serum free medium. Cells were then diluted 1:1 in 0.2% trypan blue and counted in duplicate using a Roche Cedex XS Analyzer. To analyze the recovery of cells after drug removal, medium was aspirated from UNC2025 treated cell cultures and fresh medium with or without UNC2025 was applied, and the cultures were incubated for an additional seven days prior to counting as described above.

Mycobacterium avium strain 104 (MAH 104), a clinical isolate previously described [21 (link)], was grown in 7H9 Middlebrook liquid or on 7H10 Middlebrook agar supplemented with oleic acid-albumin-dextrose-catalase (OADC; Hardy Diagnostics, Santa Maria, CA, USA) for approximately 10 days at 37 °C.
THP-1 monocytes (ATCC, TIB-202) were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS; Gemini Bio, Sacramento, CA, USA), L-glutamine, and at 37 °C in an atmosphere of 5% CO₂. THP-1 cells were treated with 20 ng/mL of phorbol myristate acetate (PMA; Sigma-Aldrich, Saint Louis, MO, USA) overnight to differentiate them into macrophages. Monolayers of 5 × 10⁵ cells/well of 48-well tissue culture plate were infected with a multiplicity of infection (MOI) of 1. After 1 h of infection, cells were washed with hank’s balanced salt solution (HBSS; VWR, Visalia, CA, USA) three times and replenished with fresh RPMI-1640 medium. At selected time-points cells were lysed, and intracellular bacterial number were determined by counting the colony forming units (CFUs) on 7H10 Middlebrook agar plates and comparing to the original inoculum used for the infection.

Unless not indicated otherwise, all chemicals were purchased by Sigma Aldrich (Darmstadt, Germany) and were of the highest purity available. MilliQ quality water was used to prepare all home-made solutions (Millipore, Darmstadt, Germany), Hank's balanced salt solution with Ca²⁺ (HBSS⁺)(1.26 mM CaCl₂, 0.49 mM MgCl₂ x 6H₂O, 0.41 MgSO₄ x 7H₂O, 5.33 mM KCl, 0.44 KH₂PO₄, 4.12 NaHCO₃, 137.93 mM NaCl, 0.34 mM Na₂HPO₄, 5.56 mM D-glucose) was purchased by Life technologies. Working solutions of tert-butylhydroperoxide (t-BuOOH) were prepared by diluting 1 M stock in HBSS⁺. Stock solutions of 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO) (50 mM) were prepared in 10 mM NaOH and kept on ice until use. Stock solutions of nitrosated cysteine (CysNO) were prepared as described (Cortese-Krott et al., 2012a (link)). Briefly, 200 mM stock solution of nitrite (VWR, Darmstadt, Germany) and acidified L-cysteine hydrochloride were mixed in equal part, equilibrated to neutral pH, kept on ice in the dark until use and diluted to final concentrations in HBSS⁺.

The procedure was adapted from Kondo et al. (2011) [11 (link)] and modified as follows. Tumor specimens were washed in HBSS (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), minced with forceps, and digested with LiberaseTM DH [12 (link)] for 2 h at 37 °C. Digested tissue was centrifuged (300× g, 5 min), washed with HBSS and filtered through a stainless 500 μm steel mesh (VWR). The flow-through was again filtered through a 40 μm cell strainer (Corning, Corning, NY, USA). The filtrate containing the TIL fraction was resuspended in Advanced RPMI 1640 (Gibco) supplemented with 2 mM Glutamine (Gibco), 1% MEM Vitamins (Gibco), 5% human serum (Sigma-Aldrich, St. Louis, MO, USA) and 100 μg/mL primocin (Invivogen, San Diego, CA, USA). IL-2 (100 U/mL), IL-7 (10 U/mL) and IL-15 (23.8 U/mL) (Peprotech, East Windsor, NJ, USA) were freshly added to culture media. For expansion, CD3/CD28 dynabeads were added (Milteny Biotech, Auburn, CA, USA). PDM, held back by cell strainer, were washed in HBSS and cultured in suspension in StemPro^® hESC SFM (Gibco) supplemented with 8 ng/mL FGF-basic (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1.8% BSA (Gibco) and 100 μg/mL primocin (Invivogen) within cell-repellent culture dish (60 × 15 mm) (Corning).

Percoll was obtained from VWR (Mississauga, ON, Canada) and density gradients were prepared in Dulbecco’s phosphate buffered saline (DPBS) and HBSS at a concentration of 65% (v/v). Recombinant human eotaxin-1 (CCL11) was obtained from Reprokine (Tel Aviv, Israel). NH₄Cl lysis buffer used during granulocyte isolation contained (in mM): 155 NH₄Cl, 10 KCO₃ and 0.1 ethylenediaminetetraacetic acid (EDTA), pH 7.2. All reagents were dissolved in RPMI 1640 Media, which sometimes contained 10% FBS and penicillin/streptomycin (1000 IU) Fisher Scientific, Walkersville, MD, USA), or in (Ca²⁺-free) HBSS (Gibco, Grand Island, NY, USA), pH 7.2–7.4. DPBS, McCoy’s 5A Medium and fluo-3 AM were obtained from Invitrogen (Burlington, ON, Canada). CDP-323 (Zaurategrast) was obtained from Adooq Bioscience (Irvine, CA, USA). Recombinant Arg-Gly-Asp (RGD) tripeptide, rat tail collagen (Type I), and bovine fibronectin were obtained from Sigma Aldrich (Oakville, ON, Canada). MACS buffer used during eosinophil enrichment was prepared in 1× DPBS and contained the following (in millimoles): 2 EDTA and 0.5% BSA (w/v).