Pvdf durapore centrifugal filter

Quantification of concentrations of chemokines was performed using the validated Luminex bead-based assay from R&D Systems (Minneapolis, MN) according to the manufacturer’s instructions. Briefly, a 20-mm-long colon tissue sample was placed into 1 mL PBS, and they were mechanically disrupted. The mixture was then centrifuged at 20,124 × g for 5 min three times and the supernatant was transferred to a 0.22 μm PVDF DuraPore centrifugal filter (EMD Millipore, Billerica, MA) to remove any particles from the solution. Filtered samples were then incubated with a capture bead cocktail on a 96-well plate in the dark for 2 h at room temperature. After incubation, wells were washed with wash buffer, incubated with a biotin antibody cocktail for 1 h next before another round washing, and incubated for another 30 min with Streptavidin-PE. Following washes, the plate was read using a Luminex 200 apparatus (Applied Cytometry Systems, UK) and analyzed with StarStation V.2.3 (Applied Cytometry Systems, UK). Total protein concentration of filtered samples was measured using Bio-Rad Protein Assay (Bio-Rad Laboratories). Results were normalized against the amount of total protein extracted from the colon tissues or the weight of the colon tissue.

Quantification of concentrations of cytokines was performed using the validated Luminex bead-based assay from R&D Systems (Minneapolis, MN) according to manufacturer’s instructions. Briefly, a 20-mm-long colon tissue sample was placed into 1 mL PBS, and they were mechanically disrupted. The mixture was then centrifuged at 10,000 rpm for 5 min three times and the supernatant was transferred to a 0.22 μm PVDF DuraPore centrifugal filter (EMD Millipore, Billerica, MA) to remove any particles from the solution. Filtered samples were then incubated with capture bead cocktail on a 96-well plate in the dark for 2 h at room temperature. After incubation, wells were washed with wash buffer, incubated with a biotin antibody cocktail for 1 h next before another round washing and incubation for another 30 min with Streptavidin-PE. Following washes, the plate was read using a Luminex 200 apparatus (Applied Cytometry Systems, UK) and analyzed with StarStation V.2.3 (Applied Cytometry Systems, UK). Total protein concentration of filtered samples was measured using Bio-Rad Protein Assay (Bio-Rad Laboratories). Results were normalized against the amount of total protein extracted from the colon tissues.