That's normally done by adding a cocktail of six enzymes to a solution containing the plants.
After five days, immature DCs were collected, transferred to new plates, and induced to mature by adding a cocktail consisting of 150ng/ml IL-6, 5ng/ml TNF, and 5ng/ml IL-1β (R&D Systems) and 1 μg/ml PGE2 (Sigma).
Non-specific proteolysis that could remove the protein signal was prevented by adding a cocktail of protease inhibitors to the buffers.
Slides were prepared for 4°C overnight incubation (12 hours) by adding a cocktail of MAP-Tau primary antibody (1 750) plus a wide-spectrum rabbit anti-cow cytokeratin antibody (Z0622; DAKO, Carpinteria, CA) diluted 1 100 in BSA/1X TBS-T.
Molecular weight standards were generated by adding a cocktail of antibodies to P190 (190 kD), Adaptin beta (106 kD), STAT-3 (92 kD), PTP1D (72 kD), Mek-2 (46 kD), RACK-1 (36 kD), GRB-2 (24 kD) and Rap2 (21 kD) to lane 40 of gels A-D.
Molecular standards for gel E were generated by adding a cocktail of antibodies to Exportin-1/CRM1 (112 kD), MCM (83 kD), Nucleoporin p62 (62 kD), α-tubulin (55 kD), Actin (42 kD), KNP-1/HES1 (28 kD) and NTF2 (15 kD) to lane 16, and antibodies to p190 (190 kD), Hip1R (120 kD), Transportin (101 kD), Calreticulin (60 kD), Arp3 (50 kD), eIF-6 (27 kD) and Rap2 (21 kD) to lane 17.
Mature DC controls were obtained by adding a cytokine cocktail as previously described [ 28].
On day 6 of culture, DC maturation was induced by adding a cytokine cocktail consisting of 10 ng ml−1 TNF α, 10 ng ml−1 IL-1 β, 1000 IU ml−1 IL-6 (R&D Systems), and 1 μg ml−1 PGE2 (Cayman Europe, Tallin, Estonia).
The conditioned media of serum-free cell cultures were cooled down on ice, centrifuged (200 g, 10 min) and passed through 0.2 μm pore filters to remove cellular debris, protected from proteolytic digestion by adding an inhibitor cocktail (7 nM pepstatin, 85 μg/ml PMSF and inhibitor cocktail complete™ Roche, Mannheim, Germany), and concentrated by ultrafiltration (Centriplus YM-3, Millipore).
Depolymerization of pretreated biomass is achieved by adding an enzyme cocktail which degrades the polysaccharides to pentoses (xylose and arabinose) and hexoses (glucose, mannose and galactose).
PCR was performed by adding a 30-μL cocktail containing D1 consensus primer, PCR buffer, and EXPAND polymerase (Roche) to the 20-μL RT reaction.
