Tris hcl

The in vitro experiments required hemolymph collection. To collect the hemolymph, the larvae were selected according to the following criteria: last instar, weighing 240–300 mg, and presenting with clear, uniform color without dark spots or grayish marks. To collect the hemolymph, we held the larva in its ventral position, punctured one of the central prolegs and collected the hemolymph. Adipose tissue and any liquid of a dark color were discarded. The collected hemolymph of ten larvae was transferred to a microtube containing 900 μL of IPS (insect physiological saline: 150 mM sodium chloride (Promega, USA), 5 mM potassium chloride (Promega), 10 mM Tris HCl (Promega) pH 6.9, 10 mM EDTA (Promega) and 30 mM sodium citrate (Sigma-Aldrich, USA) plus 10 mM N-ethylmaleimide (Sigma-Aldrich) (anticoagulant). Tubes were placed on ice, and sample collection was carried out immediately to avoid cell melanization. After centrifugation at 2000 rpm and at 4 °C for 5 min, the supernatant was discarded, cells were washed with 500 μl of cold IPS and the contents of two tubes (or several, depending on the number of assays) were pooled together in a new tube. A second centrifugation was performed under the same conditions. The supernatant was again discarded, and the cells were resuspended in 1000 μl IPS. The number of cells was determined with a Neubauer chamber.

Cellular ALP activity as an early marker of osteogenic differentiation was assessed at day 7. Cells were washed twice with PBS and then lysed with protein lysis buffer containing 50 mM Tris–HCl pH 7.4 (Promega, Madison, WI, USA), 150 mM NaCl (Sigma-Aldrich, St. Louis, MO, USA), 1 mM EDTA (Sigma-Aldrich, St. Louis, MO, USA), and 1% NP-40 (Sigma-Aldrich, St. Louis, MO, USA). ALP activity was determined colorimetrically by incubating protein lysates with the substrate p-nitrophenyl phosphate (Sigma-Aldrich, St. Louis, MO, USA) in a 96-well plate at 37°C for 30 min. The absorbance was measured at 405 nm and normalized against the corresponding protein concentrations. The values were expressed as fold change relative to undifferentiated cells.