4710 series

The nanovaccine was prepared by double emulsion method. In brief, 100 µL RNA (6 µg µL⁻¹) and 92 µL PLL (10 µg µL⁻¹) were mixed and placed on ice for 30 min to form RNA particles. Then, 100 µL tumor cell membrane fragments with the protein concentration of 6 µg µL⁻¹ and 8 µL ultrapure water were added into the above solution, and the mixture was considered as the water phase solution. 100 mg PLGA and 4 mg Ce6 were dissolved in 1 mL dichloromethane, forming the oil phase solution. To prepare the nanoparticles, the water phase (300 µL) was added into oil phase (1 mL), and then the mixture was sonicated by ultrasonic homogenizer (Cole Parmer 4710 Series) with 50% cycle duty for 60 s. Then, the mixture was poured into 2.5 mL PVA water solution (20 mg mL⁻¹) and sonicated for another 60 s. At the end of sonification, the above mixture was added to 40 mL PVA solution (5 mg mL⁻¹) and stirred for 4 h at room temperature to evaporate organic solvent. The nanoparticles were collected by centrifugation at 12 000 × g for 15 min and washed three times with ultrapure water. The obtained nanoparticles were resuspended using 4% trehalose, followed by lyophilization (SCIENTZ‐18N) for 48 h. For the controls, the RNA or cell membrane was replaced by water of the same volume.

Cellsgrown in 6 well plates were harvested (~10⁶) at 6 h following irradiation of groups treated with vehicle, 4-OHT (post-irradiation, 15 μM, 4 h), HYP (2 μM, 4 h) or 4-OHT (post-irradiation) + HYP. The lysates were sonicated in a 4710 series Cole-Palmer ultrasonic homogenizer (100% duty cycle) and boiled for 5 min at 95 °C. Appropriate amounts of lysates (15 μL maximum) were loaded onto Criterion™ TGX™ precast gels (Bio-Rad Laboratories Inc.) and ran at 200 V on ice. The proteins were subsequently transferred from the gels onto nitrocellulose membranes, using a Trans-Blot® Turbo™ transfer system (Bio-Rad Laboratories Inc.). The membranes were subsequently washed with TTBS and blocked with 5 w/v skimmed milk for 1 h at RT. The membranes were then incubated overnight at 4 °C with the selected primary antibodies, diluted according to their manufacturers’ recommendations in 5 w/v skimmed milk. The membranes were then washed three times in Tween-Tris-buffered Saline (TTBS, 5 min per wash) and incubated with the secondary antibodies according to the suppliers’ indications. The membranes were again washed three times with TTBS and incubated for 5 min with LumiGlo (KPL, Kirkegaard & Perry Laboratories, Inc.). The western blots were read at ChemiDoc™ MP Imaging System (Bio-Rad Laboratories Inc.); γ-tubulin was in all cases used as a loading control.

Two types of extraction media were used to isolate pigments from algal cells: 90 % acetone solution with respect to chlorophylls and carotenoids (Parsons et al. 1984 ) and extraction medium consisted of 0.25 M Trizma Base, hydrated 10 mM disodium EDTA (2H₂O), and 2 mg cm⁻³ lysozyme; the initial pH 9 was adjusted to final 5.5 (HCl)—in case of extraction phycobiliproteins from cyanobacteria cells (according to Steward and Farmer 1984 ). Chlorophylls and carotenoids were extracted by mechanical grinding and sonication (2 min, 20 kHz, Cole Parmer, 4710 Series) in the darkness conditions at 4 °C for 2 h. Procedure of isolation of phycobiliproteins from cells was based on combination of a gentle mechanical grinding and enzymatic (lysozyme) reaction in order to successfully disintegrate cell walls and improve pigment extraction efficiency in darkened room conditions. Filers were then incubated at 37 °C for 2 h in a dry block heat bath (Thermoleader, Uniequip) and after that kept in dark at 4 °C for 24 h. The extract was then centrifuged (20 min, 5 °C, 3210×g, Beckman, GS-6R) to remove the filters and cellular debris.
The clarified extracts were then subjected to the chromatographic analysis in case of chlorophylls and carotenoids and spectrofluorometric measurements in case of phycobilins.

The pellet providing from centrifugation at 100,000 g was treated with lysozyme for 10 min and then suspended in 150 μL of Laemmli buffer (0.1% β-mercaptoethanol, 0.0005% Bromophenol blue, 10% glycerol, 2% SDS, and 63 mmol.L⁻¹ Tris-HCl, pH 6.8). The samples were sonicated three times for 5 s each using a Cole Parmer 4710 series ultrasonic homogenizer. One hundred micrograms of the extract was subjected to SDS-PAGE and Western blotting as described above using polyclonal antibodies against XfYgiT, XfMqsR, and XfPal.