Rpmi 1640 buffer

The colon and part of the cecum were opened lengthwise and cut into 2- to 4-cm pieces, collected in 15 ml of ice-cold 1× RPMI 1640 buffer (Gibco) in a 50-ml Falcon tube, and cleaned with 20 ml of ice-cold 1× Dulbecco’s phosphate-buffered saline (DPBS; Gibco) in another 50-ml Falcon tube. The tissue was then placed into 15-ml conical centrifuge tubes filled with 10 ml of ice-cold dissociation reagent 1 (30 mM EDTA, 1.5 mM dithiothreitol [DTT], diluted into 1× DPBS) and placed on ice for 20 min. Tissues were then placed into a 15-ml conical centrifuge tube filled with 6 ml of warm (37°C) dissociation reagent 2 (30 mM EDTA, diluted into 1× DPBS) and incubated for 10 min at 37°C. After this incubation, tubes were shaken vigorously for 30 s to detach the epithelium from the basement membrane, for a total of about 80 to 90 shake cycles. Remnant intestinal tissue was removed, and the pellet cell solution was centrifuged at 800 × g for 5 min at 4°C. The supernatant was removed, and the cell pellet was resuspended in 1 ml of Tri reagent (Molecular Research Center) for subsequent RNA extraction or in radioimmunoprecipitation assay (RIPA) buffer for metabolism analysis.

The colon and part of cecum were opened lengthwise and cut into 2 to 4 cm pieces, collected in 15 mL of ice-cold 1× RPMI 1640 buffer (Gibco, Waltham, MA, USA) in a 50 mL Falcon tube and cleaned with 20 mL of ice-cold 1× Dulbecco’s phosphate-buffered saline (DPBS; Gibco) in another 50 mL Falcon tube. The tissue was then placed into 15 mL conical centrifuge tubes filled with 10 mL of ice-cold dissociation reagent 1 (30 mM EDTA, 1.5 mM dithiothreitol (DTT), diluted into 1× DPBS) and placed on ice for 20 min. Tissues were then placed into a 15 mL conical centrifuge tube filled with 6 mL of warm (37 °C) dissociation reagent 2 (30 mM EDTA, diluted into 1× DPBS) and incubated for 10 min at 37 °C. After this incubation, tubes were shaken vigorously for 30 s to detach the epithelium from basement membrane, for a total of about 80 to 90 shake cycles. Remnant intestinal tissue was removed, and pellet cell solution was centrifuged at 800× g for 5 min at 4 °C. Supernatant was removed, and the cell pellet was re-suspended in radioimmunoprecipitation assay (RIPA) buffer for metabolism analysis.