Specord s600 spectrophotometer

UV–VIS measurements were performed using a Specord S600 spectrophotometer, associated with Win Aspect software version 2.3 (Analytik Jena AG, Jena, Germany).
Solutions of LPSQ/POSS-triazole-Py and LPSQ/POSS-amide-Py at different concentrations (c_Py = 10⁻⁸ − 10⁻³ mol/L) were prepared in THF, CHCl₃ and DMF and placed in 10 mm path length quartz cuvettes. Measurements in solid state were carried out for thin films spin coated on quartz plates from solutions of LPSQ/POSS-triazole-Py and LPSQ/POSS-amide-Py in chloroform (c_Py = 10⁻⁶ mol/L).

Absorbance spectra and kinetics of carotenoid transfer between CTDH and HCPs were measured in a Specord S600 spectrophotometer (Analytic Jena) at 23 °C. To study the carotenoid transfer from holo-proteins to apo-proteins, holo-HCPs to apo-CTDHs (1 holo-HCP per 2.5 apo-CTDHs molar ratio) and holo-CTDHs to apo-HCPs (1 holo-CTDH to 5 apo-HCP molar ratio) protein mixtures were incubated in 40 mM Tris-HCl buffer (pH 8) at 23 °C for 1 h in darkness. A triplicate of absorbance spectra were recorded for 1 h and carotenoid transfer was followed by changes in absorbance at 600 nm. To determine the percentage of carotenoid transferred, a spectral deconvolution was performed using Excel to fit the data to the sum of the reference spectra of the holo-proteins involved in the experiment (described in ref. ^{26 (link)}). To study the carotenoid transfer from membranes to HCPs and CTDHs, 12 µM apo-dimers were incubated with an E. coli canthaxanthin-containing membrane suspension (48 µM canthaxanthin, measured by acetone extraction) at 33 °C for 1 h in darkness. Holo-protein formation was measured by absorbance spectroscopy after precipitation of membranes. The percentage of holo-protein formed was determined by comparing the spectra of 100% holo-proteins (at 12 µM) to those of the supernatant.

Neutral red uptake is a common quantitative assay to determine cell viability and toxic effects of the compounds in vitro. This method was used to examine larval viability in freshly isolated larvae and in larvae after 72 h of incubation with 5 μM and 50 μM of SB, SCH, DHSB as well as in untreated controls under both types of conditions. In the assay we used the In vitro Toxicology Assay Kit Neutral Red Based (Sigma-Aldrich, Darmstadt, Germany) with some modifications. Briefly, larvae were transferred from flasks into 1.5 mL tubes (in triplicates/group) and filled with 1.0 mL of fresh RPMI medium containing 20 μg/mL of neutral red. Incubation involved mild shaking at 37 °C and was terminated after 60 min. After washing the larvae, they were homogenized in 1.5 mL of solubilization solution. The supernatant containing neutral red was collected into another tube after centrifugation. The optical density (OD) of the samples was measured at 540 nm in a Specord S600 spectrophotometer (Analytic Jena, Jena, Germany). Concentrations of neutral red expressed as OD for the actual amount of larval tissue were re-calculated for 50 mg of tissue in each sample, and these values were used to calculate the viability of larval cells (in %) relative to untreated control samples (100%) at each time point.