Cellquest software package

Ten batches of BCG vaccines were diluted in PBS (1:100) and incubated at 37 °C for 24 h. Each batch’s live-cell count was measured by flow cytometry (BD FACSCanto™ II, BD Life Sciences, CA, USA) equipped with 488 nm-laser excitation and BD CellQuest™ software package (BD Life Sciences, CA, USA). Side scatter (SSC) was used for microbial cells. Overall, 50,000 events were acquired for each sample. Combined parameter of FSC-H, SSC-H and FL2 was used to gate the cells and the counting beads, and then sequential gating was done using FL1 vs. FL3 dot plots of bacteria stained with TO and PI. TO fluoresce primarily in FL1 and FL2; PI fluoresces primarily in FL3. BD FACSDiva Software was used for data acquisition, data analysis and visualization (BD Life Sciences, CA, USA). Equation (4) was used to calculate the absolute count of viable cells in the sample: $$Concentrationofbacterialpopulation=N1/N2\times N3/V\times D,$$ where N1 is the number of events in a region containing cell population, N2 is the number of events in bead population, N3 is the number of beads per test (as obtained from the BD Liquid Counting Beads label), D is the dilution factor and V is the test volume.

Mice were sacrificed by exsanguination via cardiac puncture and lungs were perfused clear of blood with saline and removed from the thoracic cavity, as previously described [25] . Lung tissue-associated cells were extracted from the right lung by mincing and enzymatic digestion, as previously described [20] (link). Mononuclear cells were collected following density gradient centrifugation (400 g for 20 min) over Histopaque (Sigma, Oakville, Ontario, Canada).
Cells were immunostained with Sca-1-FITC, c-kit-PE (BD Bioscience, Oakville, ON, Canada) and VEGFR2-APC (eBioscience Inc., San Diego, CA, USA), or isotype control antibodies (40 min at 4°C), and fixed in PBS with 1% paraformaldehyde (BDH Laboratory Supplies, Mississauga, ON, Canada), and cell data (100,000 events in the lymphomononuclear region) were acquired using a FACSCaliber flow cytometer equipped with a 488-nm argon ion laser (BD Instrument Systems, Mississauga, ON, Canada). Primitive progenitor cells (Sca-1⁺c-kit⁺) and lineage-committed EPCs (Sca-1⁺c-kit⁺VEGFR2⁺) were enumerated using the Cellquest software package (BD Biosciences). The flow cytometric gating strategy are previously described in detail in [15] (link). Doyle et al., 2011 [20] (link). Absolute numbers of cells were calculated using the percentage of population positivity obtained by flow cytometery and the total white cell count.

Cells were seeded in 6-well plates and RNA interference was performed. Cells were permeabilized with 0.1% Triton-X100 and the nuclei were then stained with propidium iodide (PI) after 24-48 h. The DNA content was measured using a FACS Caliber cytometer (BD biosciences) and analyzed with the ModFit LT (Verity Software, Topsham, ME, USA) and Cell Quest software package (BD biosciences).

In order to determine erythroid differentiation of UCB-derived CD34⁺, after 72 hours the cells (1×10⁵/ml) related to each group were gathered for flow cytometric analysis. Initially, cells were washed and suspended in PBS and labeled on ice (4°C) with anti-CD71-PE and anti-CD235a-FITC (DakoCytomation, Glostrup, Denmark) for 30 min. Then cells were rewashed and resuspended in PBS (as sheath fluid) for flowcytometric analysis. Data were acquired by FACSCalibur equipped with the CellQuest software package (BD Biosciences), and finally analyzed by Flowing software (Turk University, Finland).

Lymphocyte cells were isolated from lymph nodes by manual dissociation and were suspended in Hanks buffer (Sigma, China). T cells were further purified by CD3 microBeads (Miltenyi Biotech, Germany) and counted. Splenocytes were isolated and treated with ACK red blood cell lysis buffer (Invitrogen, China) following manual dissociation, and then the similar protocol was used for isolation of as described above. Finally, lymphocytes (1 × 10⁶) were stained with FITC-conjugated mAbs against mouse CD4 or CD8 and PerCP-Cy™5.5-conjugated against mouse CD3, all purchased from BD Pharmingen (BD, USA). Next, these stained cells were analyzed using a FACS Caliber flow cytometer (BD, USA) and the Cell Quest software package (BD, USA).

Intracellular ROS levels were evaluated using a commercial detection kit ROS-ID (Enzo Life Science, Farmingdale, NY, USA). Suspensions of AC and SC were prepared in normal culture flasks and incubated for 24 h, followed by addition of 50 μM metformin 24 h prior to 5 Gy X-irradiation. After irradiation, cells were trypsinized and resuspended in RPMI, and at least 10⁵ cells were analyzed by flow cytometry using FACS Callibur (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s protocol. MFI of each sample was normalized to that of the ACs control. All data were analyzed using the CellQuest software package (BD Biosciences).