Heraeus pico 17 centrifuge

An indospicine degradation assay was used to verify if microbial degradation of the toxin was occurring throughout the fermentation. At days 5, 9 and 14 of the fermentation, 6 × 10 mL aliquots of fermenter fluid were placed into sterile Hungate tubes [54 ] and gassed with CO₂/H₂ mix prior to sealing in order to provide anaerobic growth conditions. For three of these Hungate tubes, 200 µL of Indigofera extract was added to each tube and to the remaining three tubes, 200 µL salts solution was added to each tube. Immediately after setup, 1.0 mL volumes were removed and stored at −20 °C to provide a 0 h timepoint. The Hungate tubes were then incubated at 39 °C with rocking and 1.0 mL samples taken at 0, 9, 24 and 48 h of incubation. These 1 mL volumes were centrifuged at 16,100× g for 10 min (Heraeus Pico17 Centrifuge, ThermoScientific, Waltham, MA, USA) and the supernatant transferred into a clean microfuge tube and stored at −20 °C for subsequent UPLC–MS/MS analysis of indospicine, 2-APA and 2-APAA concentrations.

Bacterial growth was measured by monitoring the optical density (OD) at 450 nm. Biomass yield was determined by weighing the dried pellet of the cells obtained after centrifugation of 1 mL at 12,000 rpm for 10 min (Heraeus Pico 17 Centrifuge, Thermofisher Scientific, MA, USA). pH of the supernatant was measured using Seven Compact pH meter (Mettler Toledo Ltd., Leicester, UK). Nitrogen in the form of ammonium ions was estimated by the phenol-hypochlorite method [21 (link),22 (link)].
Polymer was extracted from the dried biomass using the soxhlet extraction method. To remove organic soluble impurities, dried biomass was refluxed in methanol for 24 h followed by refluxing in chloroform for 48 h to extract the polymer. Polymer solution in chloroform was concentrated using a rotary evaporator. Polymer was precipitated using ice-cold methanol under continuous stirring and dried at room temperature. Polymer yield was calculated as a percentage of dry cell weight (DCW), using the formula: $\%\mathrm{DCW}=\frac{\mathrm{Polymer} \mathrm{mass}}{\mathrm{Mass} \mathrm{of} \mathrm{biomass}}\times 100$

For protein precipitation
studies, a series of 10 mg/mL lysozyme samples and ATP or TPP concentrations
ranging from 0 to 100 mM in 10 mM Tris pH 7.0 were prepared. After
mixing, the samples were left to equilibrate at room temperature for
60 days. After 2 h of incubation, 50 μL of supernatant was collected,
centrifuged at 10,000 rpm for 10 min using a Heraeus Pico 17 Centrifuge
(Thermo Fisher Scientific Ltd., U.K.), and its protein concentration
measured. This step was repeated after 60 days of incubation at room
temperature. For samples containing TPP, the protein concentration
was determined by measuring the absorbance at 280 nm using a NanoDrop
2000 (Thermo Fisher Scientific Ltd., U.K). For samples containing
ATP, the concentration was measured using the Pierce BCA Protein Assay
Kit assay (Thermo Fisher Scientific Ltd., U.K.) per manufacturer’s
protocol. A stock solution of lysozyme with a known concentration
was used to prepare the dilution series of standard samples to obtain
the standard protein concentration curve.

PMA Dye, 20 mM in H₂O (Cambridge BioSciences, Cambridge, UK), was stored at -20°C in the dark, thawed on ice and added to 2 ml clear centrifuge tubes containing 200 μl of cell suspensions to a final concentration of 20 μM, 30 μM, or 50 μM. PMA-free samples served as controls for each condition tested. Tubes were covered with aluminium foil and shaken on an orbital shaker for 5, 10, 20, 30 or 70 minutes. Samples were then exposed to light using the PMA-Lite LED Photolysis Device (Cambridge BioSciences, Cambridge, UK) for 5, 10, 20, 30 or 40 minutes. Samples were pelleted using the Heraeus Pico 17 Centrifuge at 2000xg (ThermoFisher Scientific, Loughborough, UK) for 10 minutes at room temperature prior to DNA isolation. Non-PMA treated controls allow for the total number of B. pertussis cells to be enumerated. The number of viable B. pertussis cells calculated from PMA-treated samples can be subtracted from the total number of B. pertussis cells to provide the number of dead cells in a sample.

P. lineatus were anaesthetized with an overdose of MS-222 (1:5000, pH 7.5 adjusted with NaHCO₃) 48h after ligation or sham ligation. Blood was collected using a heparinized capillary tube following caudal transection, centrifuged at 13000xg for 5min (Heraeus Pico 17 Centrifuge, Thermo Scientific) at room temperature. The hematocrit (Hct) was measured in duplicate (nearest mm) then converted to percentage of total blood volume. Gill, kidney, anterior and posterior intestine were excised and either (1) directly snap frozen in liquid nitrogen or (2) immersed in SEI buffer [sucrose (150 mM), EDTA (10 mM), imidazole (50 mM), pH 7·3] and then snap frozen and stored at -80°C. Blood sampling was done in additional sets of six individuals but the body cavities were opened by ventral incision and the whole carcasses were immersion fixed in 10% neutral buffered formalin (NBF 10%) for 24h and stored in 70% ethanol at 4°C for immunohistological analyses.

Elastin content was quantified using the Fastin™ Elastin Kit (Biocolor, UK), as per manufacturer's protocol. The total amount of collagen in each material was analyzed by hydroxyproline assay [35 (link)]. Briefly, 5 mg of each sample were hydrolyzed in 6M HCl at 110 °C overnight. The hydrolysates were then centrifuged (Heraeus Pico 17 Centrifuge, Thermo Fisher, Ireland) at 15,000 g and room temperature for 10 min and 10×, 50×, and 100× dilutions of the supernatants were prepared. One hundred ten microliters of these dilutions were transferred to a microcentrifuge tube and 176 μl of chloramine-T reagent were added. The samples were then mixed and incubated for 10 min at room temperature. After incubation, 460 μl of Ehrlich's reagent were added, the samples were vortexed (Fisherbrand™ Classic Vortex Mixer, Thermo Fisher, Ireland) for 30 s and incubated at 70 °C for 10 min. Then, 200 μl of each sample were transferred to a well of a 96-well plate and absorbance (Varioskan Flash Spectral Scanning Multimode Reader, Thermo Fisher, Ireland) was measured at 555 nm. The hydroxyproline corresponding to the elastin (1% wt/wt) was subtracted from the total hydroxyproline content. The remaining hydroxyproline amount was used to calculate the collagen content by dividing by 0.135 (13.5% wt/wt) [35 (link)].

Resistance to collagenase [35 (link)] and elastase [37 (link)] degradation was also assessed. Briefly, 5 mg pieces of each material were cut and placed into Eppendorf tubes. One milliliter of Tris–HCl buffer pH 7.40 containing 50 U/mL of matrix metalloproteinase (MMP)-8 (17101015, Gibco®, Ireland) or Tris buffer pH 8.5 containing 0.1 U/mL of elastase (E7885, Sigma-Aldrich, Ireland) was added. The samples were then incubated at 37 °C under agitation in an orbital shaker (MaxQ 4000, Thermo Fisher, Ireland) at 150 rpm for 2, 4, 8, 12, and 24 h. The solubilized portion was discarded after centrifugation (Heraeus Pico 17 Centrifuge, Thermo Fisher, Ireland) at 13,000 rpm and room temperature for 10 min and the remaining pellets where weighed after overnight freeze drying (FreeZone Plus 4.5, Labconco, Thermo Fisher, Ireland). The percentage of weight loss over time was subsequently calculated for each material and enzyme.