Ammonium chloride potassium ack buffer

Chitosan was obtained from Sigma-Aldrich (St. Louis, MO, USA), Acros Organics (Bridgewater, NJ, USA), AK Scientific (Union City, CA, USA), MP Biomedicals (Solon, Ohio, OH, USA), Spectrum Chemical Mfg Corp. (New Brunswick, NJ, USA) and Primex (Siglufjordor, Iceland). Purified, low endotoxin Chitosan (<0.01 EU/mg) was obtained from the University of Arkansas Biologics Center (UABC) (Fayetteville, AR, USA). Sodium hydroxide was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Hydrochloric acid and acetic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Unless otherwise specified, Chitosan solutions were prepared by dissolving Chitosan in 0.1 M HCl and adjusted to pH 6.0 using NaOH.
Cell culture media components including Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute 1640 (RPMI-1640), fetal bovine serum (FBS), l-glutamine and penicillin-streptomycin solution were purchased from Hyclone Laboratories (Logan, UT, USA). Ammonium-chloride-potassium (ACK) buffer used in the process of isolating bone marrow derived dendritic cells from mouse was purchased from Lonza (Allendale, NJ, USA). Recombinant murine granulocyte-macrophage colony stimulating factor (rmGM-CSF) was purchased from Peprotech (Rocky Hill, NJ, USA). Lipopolysaccharide (LPS) from Salmonella enterica serotype enteritidis was purchased from Sigma-Aldrich.

The pancreas was minced and digested in 10 ml phenol red-free Dulbecco's modified Eagle's medium (DMEM) containing collagenase P (0.5 mg/ml) (Roche, Indianapolis, IN) at 37°C on a 200 rpm shaker for 20 min. After digestion, single cells were filtered by passing through a 70 um cell strainer and suspended in FACS staining buffer. Colon lamina propria (cLP) cells were prepared as previously described. Briefly, the intestine was cut longitudinally and sliced into 0.2–0.5 cm pieces, followed by incubating in 10 ml Ca2⁺/Mg2⁺ free HBSS containing 10% FBS, 15 mM HEPES, and 5 mM EDTA at 37°C on a 200 rpm shaker for 20 min. The intestine fragments were further digested in 10 ml RPMI 1640 containing 5% FBS, Collagenase D (0.5 mg/ml) (Roche) and DNase I (0.05 mg/ml) (Sigma-Aldrich, Saint Louis, MO) at 37°C on a 200 rpm shaker for 45 min. Samples were collected by passing through a 70 um cell strainer. cLP cells were then purified on a 44/67% Percoll (GE Healthcare Life Science, Chicago, IL) gradient by centrifugation (800 x g, 20 min at room temperature). Single-cell suspensions of lymphoid organs were prepared by mechanic disruption. Red blood cells were lysated with Ammonium-Chloride-Potassium (ACK) buffer (Lonza, Walkersville, MD). Peritoneal cells were collected by flushing 1 x PBS into the peritoneal cavity.