Pierce protein concentrator

Sertoli cell-conditioned media (SCCM) were made by culturing 15 × 10⁶ NPSCs (n = 5) in 150 mm tissue culture plates in exactly 35 mL of DMEM + 10% FBS. After 24 h incubation at 37 °C and 5% CO₂, media were collected. SCCM was concentrated by placing the max volume of 20 mL of SCCM (or of DMEM + 10% FBS as a control) into Pierce™ Protein Concentrators (ThermoScientific, Waltham, MA, USA), which has a polyethersulfone filter with a molecular weight cut off at 10 K. Protein concentrator with media were centrifuged for 15 min at 5000× g per manufacturer’s instructions and concentrated SCCM was stored at −80 °C until used. Roughly 12 mL of SCCM was obtained through this method. The HCS cytotoxicity assay was performed as previously described with the following conditions. Seventy-five thousand PAECs were plated per well on a 24-well tissue culture plate and cells were cultured overnight. All media were removed and 500 μL of either fresh DMEM + 10% FBS, concentrated DMEM + 10% FBS, or concentrated SCCM was added gently to each well. The rest of the assay was performed as described above.

Overnight cultures of JE2 WT and ΔccpA were inoculated to an OD₆₀₀ of 0.05 in TSB containing 45 mM glucose. At 0 h, 3 h, 6 h, 9 h, and 12 h, 0.5 ml culture was collected and pelleted for 3 min at 15,000 rpm. Supernatant was collected and filtered through the Pierce Protein Concentrators (3,000 molecular weight cutoff; Thermo Scientific, Rockford, IL) according to the manufacturer’s instructions. Amino acid analysis was performed with a Hitachi L-8800 amino acid analyzer by the Protein Structure Core Facility, University of Nebraska Medical Center.

FRPL protein sample from P. borbori (PbFRPL) was stored at −20 °C before being buffer exchanged into 200 mM ammonium acetate (pH 6.8) by multiple rounds of concentration and dilution using Pierce protein concentrators (Thermo Fisher). The sample was then diluted to 4 µM  (monomer) immediately before the measurements. Data were collected using in-house gold-plated capillaries on a Q Exactive mass spectrometer (ThermoFisher Scientific), operated in positive ion mode with a source temperature of 100 °C and a capillary voltage of 1.2 kV. In-source trapping was set to −100 V to help with the dissociation of small ion adducts. Ion transfer optics and voltage gradients throughout the instruments were optimized for ideal transmission. Spectra were acquired with ten micro-scans to increase the signal-to-noise ratio with transient times of 64 ms, corresponding to the resolution of 17,500 at m/z = 200, and AGC target of 1.0 × 10⁶. The noise threshold parameter was set to three and the scan range used was 350 to 8,000 m/z.