Fast blue rr

As described in detail previously [16 ], to obtain sufficient individuals of each Meloidogyne species population for electrophoretic analyses, the root-knot nematode populations under study and a reference M. javanica population from olive trees sampled at Córdoba, Spain [28 ], were increased on tomato (cv. Roma) in a glasshouse at 25 ± 3°C. For that, a single egg mass of each Meloidogyne species population was placed beneath the roots of individual tomato seedling in 12-cm pots filled with sterile loamy soil. However, since no reproduction occurred in tomato plants for M. oleae sp. nov. populations, five young, egg-laying females of M. oleae sp. nov. from galled roots of wild and cultivate olives were used for isozyme phenotype analysis [esterase (Est) and malate dehydrogenase (Mdh)]. For that, females were macerated in microtubes containing 5 μl of 20% (wt/vol) sucrose, 1% (vol/vol) Triton X-100 and 0·01% (wt/vol) bromophenol blue. Electrophoresis was carried out in 7×8-cm separating (pH 8·4) and stacking (pH 6·8) homogeneous gels, 7% and 4% polyacrylamide, respectively, 0·75-mm thick, with Tris-glycine buffer in a Mini Protean II electrophoresis unit (BioRad). Gels were stained with the substrate α-naphthyl acetate for Est and with Fast Blue RR (Sigma-Aldrich) for Mdh. Band patterns and relative migration of the bands (Rm) were compared to M. javanica [29 (link)].

After 14 days of induction with differentiation media, the cells were fixed and stained with fast blue RR (Sigma) and AdipoRed (Lonza) reagents to stain for alkaline phosphatase (osteoblast) and intracellular triglycerides (adipocyte). AdipoRed and fast blue were quantified on the Tecan microplate reader by measuring fluorescence with excitation at 485 and emission at 535 nm. Fast blue absorbance was measured at 572 nm.

Cells were plated in MSC media (Lonza) at 80 % confluence into six-well dishes and incubated for 8–12 hours to allow cell attachment. After the cells were attached, the media were changed to the respective differentiation cocktails ± 100 nM tazarotene (Sigma) for 14 days in vitro (DIV). Commercially available differentiation cocktails used were StemPro® Adipogenesis, Osteogenesis, and Chondrogenesis Differentiation Kits (Life Technologies). After 14 days, cells were fixed with 4 % paraformaldehyde for 30 minutes and stained for lineage specific markers. Akaline phosphatase activity in osteoblasts was revealed using Fast Blue RR (Sigma). Adipocytes were stained for lipid accumulation with LipidTOX-Green (Life Technologies). After fixation, cells were incubated with PBS containing LipidTOX for 1 hour and then imaged. Chondrocytes were examined for aggrecan accumulation using the immunofluorescence protocol already described.

The ALP activity of MG-63 cell monocultures and co-cultures was evaluated on days 1, 3 and 6 in cell lysates (Triton X-100 0.1%, 30 min), by the hydrolysis of p-nitrophenyl phosphate (p-NPP, 25 mM, Sigma-Aldrich) in an alkaline buffer (pH 10.3, 37 °C, 1 h). The reaction was stopped with NaOH 5 M, and the product (p-nitrophenol) was measured at λ = 400 nm in a microplate reader (Synergy HT, Biotek). Results were normalized to total protein content and expressed as nanomoles of p-nitrophenol per microgram of protein (nmol/µg protein).
For ALP staining, MG-63 mono and co-cultures were fixed in glutaraldehyde 1.5% (TAAB) in sodium cacodylate buffer 0.14 M (Sigma-Aldrich) for 15 min. Fixed cultures were incubated in a filtered solution containing sodium naphthyl phosphate (2 mg/mL, Sigma-Aldrich) and Fast Blue RR in Tris buffer solution 0.1 M, pH 10 (2 mg/mL, Sigma-Aldrich) for 1 h, protected from light. Stained cultures were observed by light microscopy (Primo Vert™ Inverted Microscope, Carl Zeiss). ALP presents a brown to black staining.