Vacufuge concentrator 5301

The stability of fresh PH_(1-110)GFP NPs and dehydrated PH_(1-110)GFP NPs stored for two years at room temperature (R.T.D.) was compared. The PH_(1-110)GFP NPs were dehydrated using the vacufuge™ concentrator 5301 (Eppendorf, Germany) at a centrifugal force of 240 g at 30°C for 30 min and stored at room temperature (28-30°C) for two years. The Agilent Bioanalyzer 2100 (Agilent Technologies, USA) equipped with the Protein 230 assay kit was used to evaluate the degradation of PH_(1-110)GFP NPs R.T.D. Electrophoretic assays were performed following the manufacturer’s instructions (Supplementary Figure 2). Finally, the results were analyzed with Agilent 2100 expert software (Agilent Technologies, USA).

A polar solvent system consisting of water (including water content from sample tissues) and methanol (water:methanol ratio of 0.8:2), and a non-polar solvent system consisting of water and chloroform (water:chloroform ratio of 1:2) were used to extract metabolites from the frozen coral homogenates. This resulted in a final chloroform:methanol:water ratio of 2:2:1.8, and total solvent volumes were to 931 µL and 1172 µL for A. cervicornis and O. faveolata homogenates, respectively. Metabolites were extracted from the frozen coral homogenates using steps modified from Reference [61 (link)]. Firstly, the pre-cooled polar solvent system was added to the homogenates in 1.5-mL microcentrifuge tubes and were bath sonicated with ice for 2 min. Next, the homogenate/polar solvent mixture was transferred into a glass vial containing the pre-cooled non-polar solvent system and was vortexed for 60 s. The extracts were then allowed to sit on ice for 10 min before centrifugation at 2000× g (standard acceleration due to gravity) at 4 °C for 5 min for phase separation. The polar metabolite-containing fraction was transferred using a glass pipette into a microcentrifuge tube and then dried in a Vacufuge Concentrator 5301 (Eppendorf AG, Hamburg Germany) at room temperature.

Protein spots were excised manually from the stained gels, washed several times with 200 μL water, dehydrated in 200 μl acetonitrile, and dried in a vacuum concentrator (Eppendorf® Vacufuge Concentrator 5301, Eppendorf AG, Hamburg). The gel pieces were treated with 100 mmol L^-1 ammonium bicarbonate, containing 20 mmol L^-1 DTT at 56°C for 30 min and then with 100 mmol L^-1 ammonium bicarbonate containing 55 mmol L^-1 iodoacetamide in the dark at room temperature for 30 min. Acetonitrile was added in between the treatments to dehydrate the gel pieces. Finally, the gel pieces were washed twice with 100 mmol L^-1 ammonium bicarbonate, dehydrated with acetonitrile and dried in the vacuum concentrator. In-gel digestion was carried out by incubation with 2 ng μL^-1 trypsin (sequencing grade modified, Promega Corp., USA) in 50 mmol L^-1 ammonium bicarbonate at 37°C overnight. Obtained peptides were extracted, washed with a buffer for desalting (10 mmol L^-1 ammonium phosphate, monobasic in 0.1% trifluoroacetic acid (TFA) and then loaded to a Prespotted Anchor Chip (Bruker Daltonics GmbH, Germany) targeted for MALDI-TOF analysis. The molecular masses of the tryptic peptides were determined on a Bruker Ultraflex time-of-flight mass spectrometer (Bruker Daltonics GmbH, Germany).

Snap frozen liver samples were used to analyze saturated, monounsaturated, and polyunsaturated fatty acids by ¹H NMR spectroscopy^{65 (link)}. In brief, liver samples (100 mg) were homogenized in a FastPrep-24 homogenizer (MP Biomedicals, CA) using 1.4 ceramic beads (Lysing Matrix D, MP Biomedicals) in 82% cold methanol. Following this, 66.6% chloroform was added to the homogenates and incubated for 10 min on ice. Samples were centrifuged at 14,000 × g, 10 min at 4 °C to separate aqueous and lipophilic layers which were dried separately in a Vacufuge concentrator 5301 (Eppendorf, Hamburg, Germany). Lipophilic extracts were then dissolved in deuterated chloroform containing octamethylcyclotetrasiloxane (OMS) as an internal standard. We used Bruker Advance 600 spectrometer (Bruker Biospin, Milton Canada) to acquire spectra using a standard pulse program (prnoesy 1d) operating at 600.22 MHz at 297 K (5 mm TXI Probe). Data were processed and profiled using AMIX version 3.9.14 (Bruker BioSpin). Lipid signals were integrated from the extracts relative to the OMS peak and then corrected for sample weight.

Liver samples were prepared according to previously published protocols (Wu et al., 2008 (link)). Briefly, frozen samples of roughly 100 mg were homogenized in a Fastprep-24 homogenizer (MP Biomedicals; Santa Ana, CA) using 1.4 mm ceramic beads (“Lysing Matrix D,” MP Biomedicals). Before homogenization, 400 μL cold methanol and 85 μL water were added. After homogenization, 400 μL chloroform and 200 μL water were added and the samples kept on ice for 10 min. Samples were centrifuged for 5 min at 2,000 × g at 4°. The aqueous and the lipophilic layer were collected separately and evaporated in a Vacufuge Concentrator 5301 (Eppendorf). Evaporated aqueous extracts were dissolved in 400 μL water and mixed with 200 μL buffer containing TSP and 50 μL deuterium oxide. Evaporated lipophilic extracts were dissolved in deuterated chloroform containing octamethylcyclotetrasiloxane (OMS) as internal standard (Thomas et al., 2012 (link)).

Stocks of PH_(1–110)GFP particles were stored at different conditions: 1) Room Temperature Dehydrated (RTD); 2) Room Temperature (RT); 3) 4 °C; 4) -20 °C; and 5) -70 °C. After 1, 3, 6 and 12 months of maintaining the particles in the different conditions, stock of each condition was taken and 6 mice group (n = 5) including a control group (PBS) were immunized. The RTD particles were dehydrated using a vacufuge™ concentrator 5301 (Eppendorf, Germany, cat. no. 5301) at a centrifugal force of 240 g at 30 °C for 30 min and were resuspended in PBS before being injected. In this experiment, no adjuvant was used. Blood sampling was performed for 2 months at 2-week intervals.