Deoxyribonuclease dnase 1

Dulbecco’s Modified Eagle (DMEM-F12) basal medium (containing sodium bicarbonate and L-glutamine), foetal bovine serum, deoxyribonuclease I (DNase), trypsin replacement enzyme 1X, thiazolyl blue tetrazolium bromide (MTT), sodium pyruvate powder, di-methyl sulfoxide (DMSO), temozolomide, disulfiram and copper gluconate were all supplied by Sigma Aldrich, Dorset, UK. Antibiotic-antimycotic solution (containing 10,000 units/mL of penicillin, 10,000 µg/mL of streptomycin and 25 µg/mL of amphotericin B), collagenase, trypan blue solution (0.4%), Hank’s Balanced Salt Solution (HBSS) and Minimum Essential Medium (MEM) were all supplied by Gibco, Waltham, MA, USA. Irinotecan, pitavastatin calcium, captopril, celecoxib, itraconazole and ticlopidine were supplied by LGM pharma, Florida, USA. Pronase powder was from Roche Diagnostics GmbH, Mannheim, Germany. Ficoll-paque density gradient cushions (1.077 +/− 0.001 g/mL) was supplied by GE-healthcare life sciences, Marlborough, MA, USA. Phosphate buffer saline (PBS) was supplied by Oxoid, Hampshire, UK.

Ultrapure reagent-grade water was obtained from a Milli-Q system from Millipore/Waters. Gellan Gum (Gelzan^™, Gelrite^®), glass beads, lysozyme, deoxyribonuclease I (DNase), bromophenol blue, MES hydrate and MES sodium salt were acquired from Sigma-Aldrich Co. (St. Louis, MO, USA). Tris-base, tween-20, glycine, imidazole, sodium chloride (NaCl), nickel chloride hexahydrate (NiCl₂.6H₂O) and methanol were purchased from ThermoFischer Scientific (Waltham, MA, USA). Calcium Chloride dihydrate (CaCl₂.2H₂O) and sodium dodecyl sulfate (SDS) were obtained from PanReac Applichem (Darmstadt, Germany). β-mercaptoethanol and N,N,N′,N′-Tetramethylethylenediamine (TEMED) were acquired from Merck (Darmstadt, Germany). Bis-Acrylamide/Acrylamide 40% and NZYColour Protein Marker II were obtained from GRiSP Research Solutions (Oporto, Portugal) and NZYTech (Lisbon, Portugal), respectively. All other reagents and supplies were of analytical grade.

Cryopreserved PBMC were thawed, washed in PBS containing deoxyribonuclease I (DNase, 10 μg/ml, Sigma-Aldrich). Cells were washed in PBS twice and then labeled with 0.5 μg/ml CellTrace Oregon Green 488 carboxylic acid diacetate, succinimidyl ester (OG; Life Technologies). Cells were washed once more with PBS and resuspended in R10 media containing recombinant human IL-2 (10 units/ml, obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH) [32 ]. Cells were plated in 96-well plates and incubated for 5 days in a 37°C incubator with 5% CO₂. On day 5, with the exception of the negative control (wells containing cells in media alone), cells were re-stimulated with PMA and ionomycin (described above) and treated with brefeldin A (10 μg/ml; Sigma-Aldrich) and monensin (1x, BioLegend) for 5 hours at 37°C to determine the cytokine capacity of proliferating T cells.

PBMCs (3–5 × 10⁶) were used to expand antigen-specific CD8⁺ T cells^{24 (link)–26 (link)}. Briefly, PBMCs were thawed in RF10 supplemented with 2 µg/ml deoxyribonuclease I (DNase; Sigma-Aldrich) and washed in serum-free RPMI. One third of PBMCs were pulsed for 1 h with IBV peptide pools (7 peptide pools ranging between 8 and 22 peptides per pool; Supplementary Table 1) in serum-free RPMI at 10 µM at 37*C. Pulsed cells were washed twice with serum-free RPMI, resuspended in RF10 and mixed with the remaining autologous PBMCs (also resuspended in RF10). Cultures were incubated at 37 °C/5% CO₂ for 10–12 days. IL-2 (Roche Diagnostics) was added on day 4 to a final concentration of 20 U/ml.

Cryopreserved PBMCs were thawed, washed in PBS containing deoxyribonuclease I (DNase, 10 μg/ml, Sigma-Aldrich). Cells were washed twice in PBS and then labeled with 0.5 μg/ml CellTrace™ Oregon Green® 488 carboxylic acid diacetate, succinimidyl ester (OG; Life Technologies). Cells were washed once more with PBS and resuspended in R10 media (RPMI 1640 supplemented with 10% heat-inactivated human serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine) containing recombinant human IL-2 (10 Units/mL, obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH) (50 (link)). Cells were plated in 96-well plates and incubated for 5 days in a 37°C incubator with 5% CO₂. On day 5, 75 μl of cell culture supernatant per well were removed and stored for Luminex analysis (see below). Cells were then resuspended in 250 μL R10 media. With the exception of the negative control (wells containing cells in media alone) cells were re-stimulated with PMA and ionomycin (described above) and treated with brefeldin A (10 μg/ml; Sigma-Aldrich) and monensin (1x, Biolegend) for 5 hrs at 37°C to determine the cytokine capacity of proliferating CD4 T cells.