Syringe filter

Lime juice samples were centrifuged at 5000× g for 10 min at 4 °C, filtered using syringe filters (0.2 µm, VWR International, PR, USA) and diluted ten times with Milli-Q water for analysis. A HPLC method was developed using a 1200 series HPLC system equipped with a quaternary pump and a thermostatted autosampler (Agilent Technologies, Santa Clara, CA, USA). Citric acid was quantified in triplicate using a diode array detector (Agilent 1100/1200) monitored at 210 nm and an Aminex HPX-87H ion exclusion column at a controlled temperature of 55 °C. The analytical conditions were as follows: mobile phase 0.045 N H₂SO₄/acetonitrile (94:6; v/v), flow 0.3 mL/min, and detector temperature 35 °C [23 (link)].

Media containing anti-IL8 monoclonal antibody derived from CHO DP-12 culture was thawed before clarification via sequential filtration through 0.45 μm and 0.20 μm syringe filters (VWR, Dublin, Ireland). Clarified media was then passed through a HiTrap Protein A column (GE Healthcare, Dublin, Ireland) at a flow rate of 1.0 mL/min. Using PBS, the column was washed to remove unretained material followed by elution of the mAb by 100.0 mM L-arginine (Sigma Aldrich) at pH 3.5. The pH of the eluate was neutralized (pH 7.0–7.5) by adding 0.50 M Tris buffer followed by a buffer exchange into PBS using Amicon Ultra centrifugal units with a 10 kDa MWCO membrane (Sigma Aldrich). The IgG content was determined using a Bradford protein assay; subsequently, aliquots containing 100 μg of protein were stored at −30°C pending further analysis.

Bone was obtained from adult pigs within 6 hours postmortem (Fleischerei Leopold Hödl, Vienna, Austria). Bone chips were harvested from the mandible with a bone scraper (Hu‐Friedy, Rotterdam, the Netherlands). Bone chips were washed with serum‐free Dulbecco's modified Eagle medium (DMEM) supplemented with antibiotics (Invitrogen Corporation, Carlsbad, California, USA). For ABL preparation, 5 g of wet bone chips were incubated while being stirred with 50 mL of 0.1 N HCl (10% weight/volume) at room temperature. ABL was harvested after 16 hours, centrifuged at 1200 RPM (358 g‐force) for 6 minutes and pH neutralized. After another centrifugation at 1200 RPM (358 g‐force) for 6 minutes, ABL was filtered sterile using a 0.2 μm syringe filter (VWR International, Pennsylvania, USA) and kept frozen at −20°C. The stocks were thawed immediately before each experiment. For bone‐conditioned medium (BCM) preparation, 5 g of bone chips were harvested from the mandible and incubated with 10 mL serum‐free culture medium DMEM (50% weight/volume) supplemented with antibiotics (Invitrogen Corporation). BCM was harvested after 24 hours of incubation at 37°C in a humidified atmosphere at 5% carbon dioxide. BCM was filtered sterile using a 0.2 μm syringe filter (VWR International, Pennsylvania, USA) and kept frozen at −20°C. The stocks were thawed immediately before each experiment.

HEK293T cells were transfected at 60–90% confluence. For a T25 flask, 2.5 μg viral DNA, 0.25 μg pVSVG, and 2.25 μg delta8.9 lentiviral helper plasmid were combined with 200 μL serum-free DMEM and 30 μL PEI max (Polysciences, Cat# 24765, polyethylenimine HCl Max in H₂O, pH 7.3, 1 mg/mL), and incubated for 15 min at room temperature. Then 5 mL DMEM supplemented with 10% FBS was added and mixed. Media was aspirated from the HEK293T cells and the 5 mL DNA mix was added gently to the cells. HEK293T cells were incubated for 48 hr at 37°C and then the supernatant (containing secreted lentivirus) was collected and filtered through a 0.45 μm syringe filter (VWR). Collected lentivirus was divided into aliquots in 0.5 mL sterile eppendorf tubes, flash frozen in liquid nitrogen, and stored at −80°C.