C6 analysis software

The activity of immune cells in the spleen and lung was analyzed by flow cytometry as described previously [21 (link)]. Percentages of T cells (CD4⁺, CD8⁺) and B cell (germinal center) from splenocytes and lung cells of mice were analyzed by flow cytometry 4 days post-challenge as described previously [6 ,22 (link)]. Splenocytes and lung cells (1×10⁶ cell/mL) in staining buffer (2% bovine serum albumin and 0.1% sodium azide in 0.1 M PBS) were incubated at 4°C for 15 min with Fc Block (BD Biosciences, CA, USA). For surface antigen staining, cells were incubated with fluorophore-conjugated antibodies (CD3e, CD4, CD8, B220, GL7; BD Biosciences) at 4°C for 30 min. Splenocytes and lung cells were washed with staining buffer and fixed with 4% paraformaldehyde for 30 min at 4°C before acquisition using a BD Accuri C6 Flow Cytometer (BD Biosciences). Data were analyzed using C6 Analysis software (BD Biosciences).

C26 cells (40,000/cm²) were treated with either oxaliplatin (10 µM) or 5-fluorouracil (1 µM). H₂O₂ (10 µM) was used as positive control for stress-induced cell death. All the abovementioned conditions were combined or not with 2 h SS-31 (10 µM) pretreatment. 48 hours after the treatment, the cells were collected (both supernatant and trypsinized monolayer), a 50 µL aliquot was used for cell count, and the remaining part was centrifuged, washed in phosphate buffer solution (PBS) and fixed in ice-cold 70% ethanol. For cytotoxicity and cell cycle assessments, the cells were incubated at RT in PBS containing 0.4 mg/mL DNase-free RNase and 10 μg/mL propidium iodide. Dead cells were measured according to the DNA content < 2n. The experiment was performed in triplicate for each condition. Data were collected using a BD-Accuri C6 flow cytometer (BD Bioscences, Franklin Lakes, NJ, USA) and analyzed with the C6 Analysis Software (BD Bioscences) or FCS Express 4 (De Novo Software, Pasadena, CA, USA).

The population of T cell (CD4⁺ and CD8⁺) and germinal center B cells (GC) from splenocytes of mice on day 7 after challenge infection were analyzed by flow cytometry. Briefly, 1 × 10⁶ splenocytes (each tube) in staining buffer (2% bovine serum albumin and 0.1% sodium azide in 0.1 M PBS) were incubated at 4°C for 15 min with Fc Block (clone 2.4G2; BD Biosciences, CA, USA). For surface staining, cells were incubated with surface antibodies (CD3e-PE-Cy5, CD4-FITC, CD8a-PE, B220-FITC, GL7-PE; BD Biosciences, CA, USA) at 4°C for 30 min. Splenocytes were washed with staining buffer and fixed with 4% paraformaldehyde at 4°C for 30 min before acquisition using a BD Accuri C6 Flow Cytometer (BD Biosciences, CA, USA). Data were analyzed using C6 Analysis software (BD Biosciences, CA, USA).

To determine apoptosis in splenocytes, Annexin-V and PI were stained using BD Apoptosis Detection Kit I (BD Biosciences, CA, USA). Splenocytes were collected at 7 days post-challenge. Then 1 × 10⁵ cell in binding buffer were centrifuged at 400 × g for 10 min and the supernatant was discarded. Cells were stained with 5 μl Annexin V-FITC and PI at room temperature for 15 min in the dark. The number of apoptotic cell was determined using a BD Accuri C6 Folw Cytometer (BD Biosciences, CA, USA) and analyzed with C6 Analysis Software (BD Biosciences, CA, USA).

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).

Flow cytometry was performed following the method previously described [14 (link)]. Splenocytes and lung cells acquired from the mice were individually processed to assess changes in immune cell populations. Cells were stimulated with 5 μg/mL of inactivated influenza virus for 2 h at 37 °C in complete RPMI media. Afterward, cells were washed and resuspended in stain buffer containing 0.2% bovine serum albumin (BSA) and 0.01% sodium azide in PBS for Fc-receptor blocking. Cells were incubated at 4 °C for 30 min with CD3-Cy5, CD4-FITC, CD8-PE, GL7-PE, B220-FITC, CD19-Cy7, and IgD-PE antibodies, which were purchased from BD Biosciences (San Jose, CA, USA). Afterward, cells were washed and fixed using 4% paraformaldehyde. Cells were acquired using the Accuri C6 flow cytometer and populations were analyzed by C6 Analysis Software (BD Biosciences, San Jose, CA, USA).