For this purpose, the specimens were supplemented with NaCl at a concentration of 80 100 mM before centrifugation.
For M. pneumoniae culture, throat swab specimens were vortexed, supplemented with amphotericin B and penicillin, and inoculated into SP-4 medium.
To do this we generated a set of occurrence points for each species based on our specimen records and supplemented with downloaded data from two public databases (Avian Knowledge Network (http://www.avianknowledge.net) and Ornis (museum specimen records only: http://ornisnet.org).org
To this end, MMR protein expression and coordinated promoter methylation were examined in colorectal specimens prospectively collected from LS mutation carriers (n = 55) during colonoscopy surveillance (10/2011 5/2013), supplemented with retrospective specimens.
Briefly, primary glioblastoma cells were isolated by mechanical disaggregation from surgical specimens and cultured in DMEM supplemented with 10% fetal calf serum (Biochrom), 1 mM glutamine (Biochrom), 1% penicillin/streptavidin (Biochrom) and 25 mM HEPES (Biochrom).
The specimens were rinsed twice with PBS supplemented with penicillin, streptomycin and amphotericin B, and minced well.
Specimens were then washed with PBS supplemented with 5% amphotericin-B and 5% penicillin-streptomycin and minced using a sterile scalpel in a 100 mm petri dish into small pieces, and the fragments were transferred into a clean tube filled with the enzymatic digestion solution containing 1 : 1 Ham's-F12, 0.05% Trypsin, 0.02% EDTA, and 3 mg/mL collagenase type-I.
Neurospheroid cultures were established from acute cell dissociation of human glioblastoma post-surgical specimens, and maintained in DMEM/F12 medium supplemented with B27 (Invitrogen, Carlsbad, CA, USA), EGF and bFGF (20 ng ml−1 each, Sigma-Aldrich, St Louis, MO, USA) according to the procedures described elsewhere (Galli et al, 2004; Singh et al, 2004).
Such knowledge, supplemented with information on herbarium specimens (Additional file: 1: Table S3 and S4) and species distribution (Figure 3) will be important in supporting conservation and sustainable utilization management decisions.
Dissociated OB specimens were cultured in serum-free medium supplemented with the mitogens EGF and bFGF.
After dehydration, the bone specimens were embedded without prior decalcification in methylmethacrylate supplemented with 20% dibuthylphtalaat and 0.008 g/ml Lucidol.
