Trypan blue

Spleens were aseptically isolated from ten mice from each group 4 weeks after challenge. Splenocyte suspensions were prepared in RPMI-1640 culture media supplemented with 10% FBS, after RBC lysis with red blood cell lysing buffer hybrid-max (Sigma-Aldrich, St. Louis, MO, USA), for flow cytometry analysis, antibody secreting cell (ASC) assays and cytokine analysis. Cells were stained with trypan blue (Welgene, Daegu, South Korea) and counted with a hemocytometer chamber under a microscope. Splenocytes from each animal were resuspended in staining buffer (2% bovine serum albumin and 0.1% sodium azide in 0.1 M PBS). Cells from individual mouse were separately incubated with Fc Block (clone 2.4G2; BD Biosciences, CA, USA) to block non-specific binding at 4°C for 15 min, and then stained at 4°C for 30 min with different combinations of FITC, PE, PE-Cy5, PE-Cy7 or APC conjugated anti-CD3e (145-2c11), anti-CD4 (GK1.5), anti-CD8a (53–6.7) (BD Biosciences, CA, USA). For memory T cell responses, anti-CD44 (IM7) and anti-CD62L (MEL-14) were used (BD Biosciences, CA, USA) as indicated [28 ]. For memory B cell responses, anti-CD45R/B220 (RA3-6B2), anti-CD27 (LG-3A10) and anti-IgG1 (A85-1) were used (BD Biosciences, CA, USA) [29 (link)]. Events were acquired on BD Accuri C6 Flow Cytometer (BD Biosciences, CA, USA) and data were analyzed using C6 Analysis software (BD Biosciences, CA, USA).

The skin was dissected and dermal fat was removed with scissors. The tissue was cut into small pieces with a scalpel and digested with 2.5 mg/mL collagenase type IV (Sigma, St. Louis, MO, USA) and 1 mg/mL DNase I (Promega, Madison, WI, USA) for 4 h at 37 °C. At the end of the incubation, the digested tissue was dissociated into single-cell suspensions using gentleMACS Dissociator (Miltenyi, Germany) in combination with C Tubes. Single-cell suspensions were smashed through a 70 μm nylon cell strainer (BD Falcon, Franklin Lakes, NJ, USA) and collected in a 50 mL Falcon tube. The cells were washed once with PBS + 10% FBS (1400 rpm, 10 min, 4 °C), and subsequently separated with 37%/70% Percoll (GE Healthcare, Chicago, IL, USA) gradients. Mononuclear cells were collected from the layer that was below 37% and above 70% gradient. After washing with PBS, the total mononuclear cell number was determined using a hemacytometer with 0.4% trypan blue (Welgene, Gyeongsan-si, Korea) before antibody staining.

Mice were anesthetized using ketamine and xylazine (40 mg/kg and 4 mg/kg, respectively) on day 21 or 24 after active immunization and were perfused through the left cardiac ventricle with cold phosphate-buffered saline (PBS, pH = 7.4) for 3 min to remove cells from the blood vessels. The spinal cord were removed, cut into small pieces using a scalpel and digested with 2.5 mg/ml collagenase type IV (Sigma, St. Louis, MO, USA) and 1 mg/ml DNase I (Promega, Madison, USA) for 15 min at 37°C. At the end of this incubation, the digested tissue was filtered through a 70-μm-pore cell strainer, and the cells were collected in a 50 ml Falcon tube and washed once with PBS containing 10% FBS (1400 rpm, 10 min, 4°C). The total cell suspension was then separated using a 37%/70% Percoll (GE Healthcare, Piscataway, NJ, USA) gradient, and mononuclear cells were collected from the layer between the 37% and 70% Percoll layers. After washing with PBS, the total mononuclear cell number was determined using a hemocytometer and 0.4% trypan blue (Welgene, Seoul, Korea) before staining.

PTX was purchased from Tocris (Minneapolis, MN, USA) and dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) to give the 100 mM stock solution. The PTX stock solution was stored at −70°C. SB203580, LY294002 and SP600125 were purchased from Millipore (Billerica, MA, USA), and erlotinib was purchased from LC Laboratories (Woburn, MA, USA). We purchased trypan blue from WelGENE (Daegu, Korea), and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] from MP Biomedicals, LLC (Illkirch, France). 4,6-dianmidino-2-phenylindole (DAPI) and propidium iodide (PI) were purchased from Sigma-Aldrich. Cycloheximide (CHX) was purchased from EMD Biosciences (San Diego, CA, USA), and MG-132 was purchased from Tocris. p53 siRNA and scrambled siRNA were synthesized from Genepharma (Shanghai, China), and Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). Primary antibodies against p-p38 MAPK, p38 MAPK, p-JNK, JNK, AKT, and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and the other primary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Second antibodies were purchased from Santa Cruz Biotechnology.

Mice were anesthetized using a combination of ketamine and xylazine at 40 mg/kg and 4 mg/kg, respectively, and were perfused via the left heart ventricle with cold sterile PBS for 3 min to remove PBMCs from the blood vessels. The liver was removed after perfusion, cut into small pieces by scissors and a scalpel and digested with collagenase type IV (Sigma, St. Louis, MO, USA; 2.5 mg/ml) and DNase I (Promega, Madison, USA; 1 mg/ml) for 15 min at 37 °C. Subsequently, the digested tissues were dissociated into single-cell suspensions using combination C Tubes and gentleMACS^TM dissociator (Miltenyi, Germany). The digested tissues were filtered using a 70-μm-pore cell strainer, and subsequently the cells were washed once with PBS (10% FBS). Mononuclear cells were collected from the 40/70% Percoll interphase after discontinuous Percoll gradient. The number of total mononuclear cells was determined using 0.4% trypan blue (Welgene, Seoul, Korea) and a hemocytometer before staining after washing with PBS.