Zeba 7k mwco desalting columns

Goat F(ab′)₂ anti-mouse Igκ (Southern Biotech) was biotinylated with 20-fold molar excess of NHS-LC-LC-biotin (Thermo Fisher Scientific, Inc.) and labeled with Cy3 monoreactive dye pack (GE Healthcare) in sodium carbonate buffer (biotinylated anti-κ-Cy3). Excess dye was removed using Zeba 7K MWCO desalting columns (Thermo Fisher Scientific, Inc.). Splenocytes were incubated with biotinylated anti-κ-Cy3 for 30 min on ice. Samples were incubated at 37°C for the times indicated before fixation with 2% PFA on ice for 20 min. Cells were stained with fluorescently labeled streptavidin to label the biotinylated anti-κ-Cy3 on the surface. Flow cytometry was used to determine antigen internalization defined as the reduction in the amount of biotinylated anti-κ-Cy3 remaining on the surface as a percentage of the amount on the surface of cells that had been left on ice.

Commercial lyophilized Fb powder was dissolved in 0.9% aqueous NaCl at 37 °C and transferred to buffer 1 or buffer 2 using Zeba 7K MWCO desalting columns (Thermo Fisher Scientific, Waltham, MA, USA). The protein concentration was determined using extinction coefficients E^0.1%_280nm for Fb: 1.51 [64 (link)], binase: 2.2 [9 (link)], trypsin: 1.6 [65 (link)]. Tb, at a concentration of 113 NIH units/mg, was dissolved in buffer 1. Stock solutions of Fb and Tb were aliquoted, frozen in liquid nitrogen and stored at −18 °C until use. Stock solutions of trypsin at a concentration of 1 mM, binase (14 mg/mL), and RG (10 mg/mL) were prepared immediately before measurements.
Fibrin coagulation was performed by the introduction of Tb, but without the addition of external calcium, in order to exclude the toxic effect of Ca²⁺ ions on the cells under study.

All chemicals of the highest purity were purchased from Sigma-Aldrich (St. Louis, MO) and used as received. All DNA sequences were purchased from IDT DNA Inc. as HPLC purified grade and used without further purifications. NHS ester modified fluorophores, Zeba desalting columns (7k MWCO) were purchased from Thermo Fisher Scientific. G-25 size-exclusion columns were purchased from GE Healthcare.