Pd10 sephadex g 25 desalting columns

Preparation of BSA^Ald and binding of Gd³⁺ probe to protein was carried out according to modified procedures (48 (link)). To a solution of bovine serum albumin (BSA) (100 mg) dissolved in phosphate buffered saline (4 mL, pH 7.4, 0.25 mM) was added sodium ascorbate (20 mg), ferric chloride (120 μL, 10 mM) and 20 μL H₂O₂ (30% w). The reaction was stirred at 37 °C for 24 h, and sodium ascorbate (20 mg) was added repeatedly every 8 h. After 24 h, the protein was purified using PD-10 Sephadex G25 desalting columns (GE Healthcare), eluted with PBS. Protein concentration was assessed using the ‘Micro BCA Protein Assay Kit’ (Thermo Scientific, 23235). Protein carbonyl concentration was determined by ‘Protein Carbonyl Content Assay Kit’ (Sigma-Aldrich, MAK094-1KT). BSA^Ald had an aldehyde concentration of 4 aldehyde/protein. The protein solutions were kept at a concentration of 20 mg/mL for further use.

Purified, quantified colon resection derived VLP preparations were labeled with 0.01 μM SYTO™ 16 Green Fluorescent Nucleic Acid Stain (Thermo Fisher Scientific, #S7578) according to manufacturer’s instructions. Labeled VLPs were then separated from unincorporated dye using PD10 Sephadex G-25 desalting columns (GE Healthcare, #17085101) according to manufacturer’s instructions. Uptake assays of SYTO16-labeled VLPs were performed with human peripheral blood monocyte derived macrophages. Cells were seeded at a density of 10⁵ cells/well in a 24-well plate. Cells were delivered 10⁶ or 10⁷ non-IBD, UC or CD VLPs or SM buffer control by adsorption in 100 μL for 1h, washed and left for 3 hours at 37°C for uptake analysis or 4°C for adsorption analysis. Cells were removed from the culture plate surface with cold PBS, washed in flow cytometry buffer twice, stained for live–dead discrimination and acquired on an LSR II flow cytometer (BD Biosciences). For the inhibition assays, cells were treated with 3.0 mg/mL of Brefeldin A (Invitrogen, #00-4506) for 30 min at 37°C before addition of labeled VLPs and during the adsorption. Data was analyzed using FlowJo v10 software (TreeStar).

Purified Pf4 preparations were labeled with Alexa Fluor 488-labeled TFP ester (Molecular Probes, Cat. No. A37570) following the manufacturer’s protocol. Following labeling, labeled Pf4 viral particles were separated from unincorporated dye using PD10 Sephadex G-25 desalting columns (GE Healthcare, Cat. No. 17085101) according to the manufacturer’s instructions. The conjugate was then quantitated for Pf concentration using qPCR and appropriately diluted in PBS.