For live cell microscopy mast cells (2 × 105 cells/well) were stained with Vybrant DiI Cell-labelling solution (Invitrogen) according to the manufacturer's instructions and seeded into a 35-mm glass-bottom micro- well dish (MatTek, Ashland, MA, USA).
Monocytes were isolated by CD14-positive selection using anti-CD14 magnetic beads (Miltenyi Biotec, UK), according to the manufacturer's instructions and seeded in complete medium (RPMI 10% FCS (PAA), 2 mM L-glutamine (PAA), 100 U/mL penicillin (PAA) and 100 µg/mL streptomycin (PAA)), supplemented with 50 ng/mL recombinant M-CSF (R&D Systems) for 7 days.
Following the manufacturers instructions, we seeded 1×105 cells suspended in DMEM (100 µl) onto the coated ECM-substrates and analysed for the cells ability to bind to the respective ECM proteins using a fluorescence plate reader (485/530 nm).
Total PBMC or PBMC depleted of CD25+ T cells with anti-CD25-coated microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions, were seeded at 2×106 cells/ml per well of a 24-well plate in T cell medium containing 20 IU/mL IL-2 and 10 ng/mL IL-7.
Red blood cells were lysed using BD Red Blood Cell Lysis solution (as per the manufacturer's instructions), and cells were seeded onto 96-well U-bottom plates.
MDMs were nucleofected with 5 μg of plasmid DNA using the macrophage nucleofection kit (Amaxa Biosystems, Cologne, Germany) according to the manufacturer's instructions, seeded into four-well Lab-Tek Chambered Coverglass units (Thermo Fisher Scientific) and incubated at 37°C for 24 hours.
According to xCELLigence (Roche) manufacturer's instructions, cells were seeded in duplicates into E-plates 16 (Roche, cat# 05469830001) after baseline measurement.
Provided seed multiplication instruction and support to over 1 435 progressive seed farmers belonging to 16 farmers groups through participation in 10 farmer field schools (FFS), including 11.1 tonnes of foundation seed and 600 agricultural tools.
Signed up for the Latin Scientific course, he studied the Provençal dialect with William Pierce Shephard and Old English with Joseph D. Ibbotson; with Shephard he read Dante and from this began the idea for a long poem in three parts – of emotion, instruction and contemplation – planting the seeds for The Cantos.
There are thorough instructions in "Seed to Seed" by Suzanne Ashworth (Seed Savers Exchange, 2001).
