Sigma blend collagenase type f

Samples of the total small intestine were rinsed in PBS and then treated with 2 mmol/L EDTA in PBS for 30 min to remove the epithelial cells. The residue was then digested with Sigma Blend Collagenase Type F (Sigma-Aldrich Co, Ltd., St Louis, MO) for 20 min and separated in the Percoll gradient to obtain LPMCs. In the primary cultures, 1 × 106 cells of LPMCs per well were cultured in 100 μL of 10% FCS/RPMI1640 for 24 h. At that time, 40 µL each of PMA (Phorbol 12-Myristate 13 Acetate) (Merck KGaA, Darmstadt, Germany) and lonomycin (Merck KGaA) were added to the culture medium. After culturing for 24 h, the supernatant was collected and stored in a freezer at −80 °C until it was used for ELISA. LPMCs were washed with PBS and stored in a freezer at −80 °C until they were used for mRNA expression analyses, ELISA, and flow cytometry.

Samples of the total small intestine were rinsed in PBS and then treated with 2 mmol/L EDTA in PBS for 30 min to remove the epithelial cells. The residue was then digested with Sigma Blend Collagenase Type F (Sigma-Aldrich Co, Ltd., St Louis, MO) for 20 min and separated in the Percoll gradient to obtain LPMCs. In the primary cultures, 1 × 106 cells of LPMCs per well were cultured in 100 μL of 10% FCS/RPMI1640 for 24 h. At that time, 40 µL each of PMA (Phorbol 12-Myristate 13 Acetate) (Merck KGaA, Darmstadt, Germany) and lonomycin (Merck KGaA) were added to the culture medium. After culturing for 24 h, the supernatant was collected and stored in a freezer at −80 °C until it was used for ELISA. LPMCs were washed with PBS and stored in a freezer at −80 °C until they were used for mRNA expression analyses, ELISA, and flow cytometry.