Optima tl100 centrifuge

The method for immunoisolation of exosomes described elsewhere was followed^{64 (link)}. Briefly, antibodies for immunoisolation (mouse monoclonal anti-human CD63 (NBP2-32830 0.1 mg, Novus Biologicals) and normal mouse polyclonal IgG (Millipore, Cat. No. 12-371) at a ratio of 1 μg of antibody per 100 μL of beads were coupled to Pierce Protein A Magnetic Beads (Dyna beads) by incubation at 4 °C overnight. Beads were then washed three times with 500 μL of PBS 0.001% Tween, resuspended in 500 μL of the same buffer, to which exosomes (2 × 10¹⁰) were added, followed by overnight incubation at 4 °C with rotation. Bead-bound exosomes were collected and washed three times in 500 μL of PBS-Tween. Exosomes were eluted with high-salt buffer and washed again and centrifuged at 100,000g for 1 h at 4 °C in a TLA 110 rotor (Beckman, Optima TL100 centrifuge).

Roots, leaf blades, leaf sheaths and tassels of three months maize plants and the 24 h imbibed seed embryos were frozen in liquid N₂ and kept at − 70 °C. Two grams of each tissue was crushed, and the fine powder was homogenized in buffer (50 mM Hepes/KOH pH 7.0, 300 mM sorbitol, 1 mM EDTA, 2 mM DTT, 1 mM PMSF and 1 tablet of protease inhibitor cocktail per 50 mL, Complete, Roche, Mannheim, Germany) in a ratio of 2 volumes per 1 g of tissue, using a tissue tearor homogenizer (Biospec Products, Daigger and Co. Inc. Vernon Hill, Il, USA) for 60 s. The tissue was filtered through a four-layer of pre-wet cheesecloth; the filtrate was centrifuged 5 min at 2400 g at 4 °C. The supernatant was centrifuged at 14,000 g for 10 min. The pellet contained the mitochondria fraction and the supernatant contained the cytosolic fraction. The supernatant was centrifuged at 110,000 g for 45 min in a TLA-100.4 rotor in an Optima TL-100 Centrifuge (Beckman, Palo Alto, CA, USA) at 4 °C to eliminate the microsomal fraction. The supernatant or cytosolic fraction was supplemented with 10% glycerol and stored at − 70 °C until use. The mitochondrial fraction was washed with 1 mL of homogenization buffer and centrifuged at 14,000 g for 10 min. The pellet was resuspended in 1 mL of homogenization buffer supplemented with 10% of glycerol and stored in aliquots at − 70 °C until use.