Centriprep ym 3

The Caco-2 cells were routinely grown at 37°C in a 95% air–5% CO₂ atmosphere in Dulbecco's modified Eagle medium (DMEM) with high glucose (Sigma-Aldrich Co) and 10% heat-inactivated (56°C, 30 min) fetal bovine serum (FBS; Gibco, Grand Island, NY, USA). The cells were harvested using trypsin-EDTA (Gibco) and suspended with DMEM-10% FBS to stop the reaction with trypsin-EDTA. The cells were collected from the cell suspension by a centrifugation, and were washed with Dulbecco's phosphate buffered saline (DPBS; Gibco). Then, the cells were resuspended with DMEM without any serum, and the mixture was centrifuged twice. Finally, the cellular suspension in DMEM was adjusted to 1.0 × 10⁵ cells per 75 cm² of the tissue culture flask (Thermo Scientific, Waltham, MA, USA). The culture medium was replaced every 5 days, and the supernatant collected after 5, 10, and 15 days of culture was used in subsequent experiments. The collected supernatant was filtered with Centriprep YM-3 or YM-10 (3 or 10 kDa; Millipore, Bedford, MA, USA). For some experiments, 500 μl of the various Caco-2 cell supernatant samples were treated with 25 μl of Immobilized TPCK Trypsin (Thermo Fisher) for 1 h at 37°C; the Immobilized TPCK Trypsin was then removed by centrifugation (10,000 × g for 10 min).

To harvest ESP, the standard procedure for H. contortus described by Yatsuda et al. was used [16 (link)]. Briefly, H. contortus (Nanjing strain) adult worms were harvested from the abomasum of an experimentally infected donor goat, washed several times with PBS, and incubated for 4 h in RPMI 1640 medium (100/ml) containing antibiotics (100 IU of penicillin, 0.1 mg/ml streptomycin, and 5g/ml gentamicin) at 37°C under 5% CO. The medium was then removed, and the parasites were incubated in new medium containing 2% glucose overnight. The supernatant was collected, centrifuged, filter-sterilized (0.2 m), concentrated, and desalted (10 mMTris, NaCl pH7.4) using 3 kDa filters (Centriprep YM-3, Millipore). The protein concentration was determined by the Bradford assay [31 (link)].

In addition to (FAME), the identity of Sb5 was determined by sequencing the amplicons of 16S rDNA of the isolate with primers 968F and 1410R65 (link). Both strands of the sequences were processed with BioEdit V7.2.5 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html)66 and BLASTed against 16S rDNA sequences of microbes at http://blast.ncbi.nlm.nih.gov to match to a species level. The final sequence was then deposited into GenBank.
Sb5 CFF was fractionated with centrifugal filter columns to estimate the sizes of active components. CFF was first loaded into Centriprep YM-3 (MWCO 3 kDa) and centrifuged as instructed by the manufacture (Millipore.com). The filtrate was designated as <3 kDa fraction. The remaining CFF in the column was subsequently loaded to YM-10 (MWCO 10 kDa) and centrifuged. The filtrate collected was designated as 3–10 kDa fraction while the remaining in the second column was processed with the third column, Centriprep YM-30. The resulting filtrate was designated as 10–30 kDa fraction and the solution that did not go through was designated as >30 kDa fraction. These four fractions were then tested together with the controls CFF and SDW for bioactivities in the microscopy and infection assays described above.