Vivaspin 500 concentrator

CaptureSelect FSH Affinity Matrix resin leakage was evaluated by ELISA using a CaptureSelect FSH Ligand Leakage kit (Life Technologies, Carlsbad, CA, USA). MgCl₂ at a high concentration can affect antibody interaction; hence, the eluates from the affinity column were subjected to diafiltration in exchange with TBS solution using a Vivaspin 500 concentrator (Sartorius, Goettingen, Germany) with a 3 kDa molecular weight cut-off to retain the affinity ligand, which is a 14 kDa recombinant mini-antibody. The final concentration of MgCl₂ in the samples was less than 16 mM. The samples were diluted with 1% BSA-TBS. ELISA was performed according to the manufacturer’s guidelines, with the use of placebo solution as diluent for the analysis of r-hFSH drug substance.

For ¹¹¹In‐labeling, DTPA‐conjugated CLEC2.Fc (1 mg) and DTPA‐conjugated hIgG1 (1 mg) was suspended in 200 μl of sodium acetate buffer (0.1 M, pH = 6.8), 8 mCi of 111InCl3 (INER, Taoyuan, Taiwan), respectively, and incubated at 37°C for 60 min. The mixtures were further purified by using Vivaspin® 500 concentrator (10 K MWCO, Sartorius, Goettingen, Germany) to remove the unlabeled ¹¹¹In as mentioned above. Finally, the concentrated and purified samples were responded in PBS. The specific activity of ¹¹¹In‐DTPA‐CLEC2.Fc and ¹¹¹In‐DTPA‐hIgG1 were > 4 mCi/mg, respectively. The radiochemical purities of ¹¹¹In‐DTPA‐CLEC2.Fc and ¹¹¹In‐DTPA‐hIgG1 were measured using instant thin layer chromatography (iTLC, Agilent Technologies, Santa Clara, CA); 100 mM EDTA in 100 mM ammonium acetate acts as a mobile phase (¹¹¹In‐labeled compounds Rf = 0, ¹¹¹In‐EDTA Rf = 1). The iTLC sheets were analyzed using a radioactive scanner (AR‐2000 radio‐TLC Imaging Scanner, Bioscan, France). The radiochemical purity > 90% was acceptable for the consequent use in animal studies.

To test the phagocytosis of wild type bacteria and the different pneumococci mutants, 1 × 10⁵ J774A.1 cells were seeded per well in a tissue culture coated 12-well or 24-well plate (Corning, Inc., Corning, NY) and allowed to adhere overnight. Cells were pretreated for 24 h with either PBS, LPS, IL-4, or pEVs (total of 20 μg/ml in 1 ml per 400-μl or 200-μl volume). The isolated pEVs were first concentrated to ∼60 μg/ml using 100,000 MWCO filters (Vivaspin 500 concentrators; Sartorius AG, Germany) prior to adding them to macrophages. Thereafter, cells were infected with 25 μl of pneumococcal suspension containing ∼5 × 10⁵ CFU, resulting in an MOI of 5, followed by incubation at 37°C for 30 min. Postincubation, the cells were washed three times with PBS and then incubated for either 30, 60, 120, or 240 min in RPMI culture medium containing 10 μg/ml penicillin and 200 μg/ml gentamicin to kill extracellular pneumococci. The cells were then washed five times with PBS, and phagocytosed pneumococci were harvested by treating the infected J774A.1 cells with 0.025% saponin for 15 min at 37°C. Recovered bacteria were enumerated by viable plating on blood agar plates.

Isolated plastoglobules in sucrose fractions were purified and concentrated in TrE buffer (5 mM Tricine-KOH, pH 7.5, 0.2 mM EDTA, and 0.2 mM dithiothreitol) using disposable ultrafiltration devices (Vivaspin 500 Concentrators, Sartorius Stedim Biotech GmbH, Goettingen, Germany). First, 400 µL of deterged fractions were precipitated as described previously [78 (link)], with slight modification; obtained PG proteins were resuspended in 10 µL in TrE buffer, and the BCA protein assay kit for low concentrations (ab207002, Abcam, Cambridge, UK) was used to quantify the protein content. The assay was performed according to the manufacturer’s instructions. An equal volume of every sample was added to the BCA working reagents, and the reactions were incubated for 120 min at 37 °C. Absorbance was read at 562 nm on a NanoDrop 2000/2000c Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Protein concentrations were calculated using bovine serum albumin (BSA) standards and a four-parameter logistic curve using NanoDrop BCA PROTEIN software (v. 1.6, Thermo Fisher Scientific Inc., Waltham, MA, USA).

For biotinylation of CaM, the EZ-Link Sulfo-NHS-SS-Biotin kit (Thermo Scientific) was used. CaM was concentrated via centrifugal concentration (5-kDa cut-off Vivaspin 500 concentrators; Sartorius) to a concentration of 6 mg/mL and then biotinylated according to the manufacturer’s instructions with a 20-fold molar excess of biotin reagent at 4°C overnight. Non-reacted Sulfo-NHS-SS-Biotin was removed by dialysis against 1 x PBS. Successful biotinylation was confirmed by dot or Western blotting and streptavidin-based detection described under “CaM overlay”.
The N-terminally His₁₂-tagged SNAP-tag protein (New England Biolabs) was Ni²⁺-affinity purified and covalently labeled with biotin according to New England Biolabs’ instructions.

Traut’s reagent (2-iminothiolane) was obtained from Toronto Research Chemicals (Ontario, Canada). Bovine Serum Albumin (BSA), Ellman’s reagent, and glycine were purchased from Sigma Aldrich. The PD-10 Desalting Column (Sephadex G-25 M) was purchased from GE Healthcare, and the Vivaspin 500 concentrators were purchased from Sartorius. Gold-containing solution was obtained from Transene Company, Inc.