Fp 6500 spectrophotometer

Coverslips were saved after Fmoc protected amino acid addition and after deprotection by piperidine to indicate all stages of SPPS. Coverslips were dried and mounted onto a microscope slide and fluorescence spectra recorded with a JASCO FP-6500 spectrophotometer using a technique that was specifically developed for SPPS coverslips by Zelzer et al.^{44 (link)}. Coverslips were angled a 30° from the incident light and exposed to an emission spectra = 270 nm and excitation spectra = 320 nm with a slit width of 20 nm (light source and detector).

MDA-MB-231 and SH-SY5Y cells in suspension were loaded with Fura-2 AM (5 µmol/l) through incubation in HEPES buffer solution (HBS) for 30 min at 20 °C and then for 15 min at 37 °C. A spectrophotometer was used to measure fluorescence oscillations for 300 s (FP-6500 spectrophotometer, Jasco, Tokyo, Japan). The temperature was maintained at 37 °C, and magnetic stirring was implemented during the measurements with alternating excitation wavelengths of 340 and 380 nm; the fluorescence emission was detected at 510 nm before each experiment. The 380 nm/340 nm signal was calibrated using the Grynkiewicz et al. method [23 (link)] before each experiment. Briefly, the fluorescence ratio was measured in HBS lacking Ca²⁺ supplemented with EGTA (1 mM) and in HBS supplemented with ionomycin (300 nM) containing 2 mM Ca²⁺, the Ca²⁺ concentration at which Fura-2 is saturated. Maximal and minimal ratios (Rmax and Rmin) were obtained under these two conditions, and the [Ca²⁺]_i values were derived using the following equation:
[Ca ²⁺]_i = Kd (R − Rmin/Rmax − R)(Sf2/Sb2),
R is the experimentally measured ratio, Sf2 is the fluorescence measured at 380 nm in Ca²⁺-free conditions, and Sb2 is the fluorescence measured at 380 nm with saturating Ca²⁺ (2 mM).