Ammonium chloride potassium ack buffer

Spleen and MLN cells suspension were made by cutting the spleen or MLN with scissors, followed by digestion using 1 mg/mL Collagenase D (Roche) and 50 µg/mL DNase I (Roche) at 37 °C for 45 min. The cell suspensions were filtered through a 70-μm cell strainer (BD Biosciences), red blood cells lysed by the ammonium-chloride-potassium (ACK) buffer (Thermo Fisher) at RT for 5 min and the cells were resuspended in PBS + 10% FBS for further analysis. Colons were dissected, and the stool removed by washing two times with HBSS 1X without Ca²⁺ and Mg²⁺ and finally opened longitudinally. After that, the colon was cut into 0.5 cm pieces, washed with HBSS 1X without Ca²⁺ and Mg²⁺ and incubated in IEL medium (IMDM containing 2% FBS and 1 M HEPES) at 37 °C for 30 min, while constantly stirring. Colon pieces were washed using a 70-μm cell strainer, followed digestion using 1 mg/mL Collagenase D (Roche) and 0.25 mg/mL DNase I (Roche) in IEL medium at 37 °C for 45 min while constantly stirring. Digested tissue was passed through a 70-μm cell strainer obtaining a single cell suspension that was subjected to centrifugation in a Percoll gradient (67%/44%). Mononuclear cells were removed from the interphase and resuspended in culture medium for further analysis.

Blood of immunized rabbits was used for peripheral blood mononuclear cell (PBMC) isolation through Ficoll separation. In short, blood was diluted 1:1 with phosphate-buffered saline (PBS), loaded on a Ficoll layer, and centrifuged for 30 min at room temperature at 400 × g with an acceleration speed of 7 and a deceleration speed of 0. PBMCs were isolated, washed with PBS, and subsequently resuspended in 1 to 2 ml of ammonium-chloride-potassium (ACK) buffer (Thermo Fisher Scientific) to remove red blood cells. PBMCs were counted, suspended in fetal calf serum with 10% dimethyl sulfoxide (DMSO), and directly frozen at −150°C.