Hi 96801 refractometer

A refractometer (HI 96801 Refractometer, Hanna Instruments Inc., Woonsocket, Rhode Island), was used to determine sucrose levels of fruits by measuring the refractive index to determine the % Brix of sugar in an aqueous solution (where one degree Brix equals 1 g of sucrose in 100 g of solution). To get aqueous solution, fruit parts (exocarp or mesocarp) were placed in a 2 mL microtube (14217394, Axygen, Corning, NY) and a tissue grinder was used to crush the fruit parts. A plastic pipette was then used to collect a sample of this solution and place it onto the prism surface of the refractometer. Ten randomly selected fruits were used for such measurements at each developmental stage of each species, with three replicates of each type (exocarp and mesocarp). These three samples were collected from different positions on each sample fruit and averaged within fruits.

Turbidity measurements was made using a 2100P ISO Turbidity meter (Hach Co., Loveland, CO, USA). Refractive index (RI) was measured on an HI96801 Refractometer (Hanna Instruments Inc., Woonsocket, RI, USA), in Brix. pH was measured using a HI8424 pH meter (Hanna Instruments Inc., Woonsocket, RI, USA), and conductivity was measured using a WTW Conductivity Meter LF95 (Christian Berner AB, Partille, Sweden).

LR were prepared using a previously described protocol with minor alterations [11 (link)]. Briefly, cells grown to confluence in 60 × 15 mm dishes and treated for 72 h with NB-DNJ at concentrations of 50 or 100 µM. All procedures were performed on ice or at 4 °C. Cells (2 × 10⁶) were solubilised in 1% Lubrol in 50 mM Tris, 150 mM NaCl, pH 7.6 and 50 µL protease inhibitors. Homogenisation was performed by passing the cell lysate 20 times through a 21 G needle. The lysates were then centrifuged at 1500× g for 20 min at 4 °C and the supernatant loaded onto a discontinuous sucrose gradient as described before. Gradients were centrifuged at 4 °C for 18 h at 100,000× g using a Beckman SW40 rotor. Ten fractions were collected on ice from top to bottom, with fractions 1–3 containing predominately the LRs fractions, and 8–10 containing predominately the detergent-soluble cell fraction. The sucrose content was quantified from each gradient fraction by using a HI 96801 refractometer (HANNA instruments). The quality and location of LR was assessed by Western blotting of the gradient fractions using anti-FLOT2 antibody (1:5000; sc-28320 Santa Cruz, 200µg/mL, Santa Cruz, California USA).