2.5g l trypsin 1mmol l edta solution

The murine intestinal epithelial MODE-K cell line (sex: female) was kindly provided by Prof. Richard Blumberg (Harvard Medical School, Boston, USA). MODE-K cells were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque) supplemented with 10% FBS, $2\text{\hspace{0.17em}mM}$ of glutamine, $100\mathrm{U}/\text{ml}$ penicillin, and $100\mathrm{\mu }\mathrm{g}/\mathrm{mL}$ streptomycin, in an atmosphere of 95% air and 5% ${\mathrm{CO}}_2$ .^{70 (link)} Cells were spread in 96-well plates at $1\times {10}^4$ cells/well, and $\mathrm{1,000}\mathrm{\mu }\mathrm{g}/\mathrm{L}$ of MP, $200\mathrm{\mu }\mathrm{M}$ of palmitic acid (PA),^{33 (link)} and $50\text{\hspace{0.17em}ng}/\mathrm{mL}$ of recombinant mouse IL-22 protein (NBP2-35122, Funakoshi) were added for 24 h on day 5.^{71 (link)} After 24 h, the cells were then washed twice with cold PBS, detached with $2.5\mathrm{g}/\mathrm{l}\text{-Trypsin}/1\text{\hspace{0.17em}mmol}/\mathrm{l}\text{-EDTA}$ solution (Nacalai Tesque), and centrifuged at $300\times g$ for 5 min at 4°C, and the supernatant was discarded. RT-PCR was performed as described in the “Gene Expression Analysis in Murine Jejunum and Liver” section, and the mRNA expression level of Muc2 was quantified ( $n=6$ ). To evaluate the intracellular accumulation of MPs, the pellet was counted after centrifugation. Briefly, $1\times {10}^4$ MPs were fractionated and then diluted into $200\mathrm{\mu }\mathrm{L}$ of PBS. The cell lysate was transferred to a black 96-well plate, and luminescence was measured using an Orion L microplate luminometer (Berthold Detection Systems) ( $n=6$ ). Signals from cells without MPs, PAs, and IL-22 were assigned a relative value of 1.0.