Facsaria 2 flow cytometry

The percentage of apoptosis was detected by flow cytometry after cells were stained with Annexin V and PI. The Annexin V/PI Apoptosis Detection Kit was purchased from Yeasen, China and used according to the manufacturer's guidelines. Briefly, cells in various groups were harvested, washed three times with PBS, resuspended in 1× binding buffer and stained with the Annexin V and PI solution for 30 minutes at 37°C without light. Next, the cells were sent for testing by BD FACSARIA II flow cytometry (Becton Dickinson).

Flow cytometry analysis was conducted by FACS Aria II flow cytometry (BD Bioscience, USA). For surface staining, suspensions of PBMCs were stained on ice using predetermined optimal concentrations of each antibody for 30 min, and fixed using fixation buffer (BD PharMingen, USA). Tregs identified with CD4⁺CD25⁺CD127⁻ expression were stained with human regulatory T cell Cocktail (BD PharMingen, USA) [20 (link)] and Bregs identified with CD19⁺CD24^hiCD27⁺ expression were stained with human anti-CD19, human anti-CD24, and human anti-CD27 (BD PharMingen, USA) [21 (link)]. Intracellular IL-10 analysis was performed by flow cytometry, as described previously [22 (link)]. Briefly, cells were resuspended (2 × 10⁶ cells/ml) in medium and stimulated with ODN2006 (10 μg/ml; Sangon Biotech, Shanghai, China) for 24 hrs with leukocyte activation cocktail (2 μl/ml; BD GolgiPlug™, BD Pharmingen, USA) added during the final 5 hrs before staining. After surface staining, cells were fixed, permeabilized using a Cytofix/Cytoperm™ Kit (BD PharMingen, USA), and stained with human anti-IL10 (BD PharMingen, USA) according to the manufacturer’s instructions. Results are expressed as frequency of Tregs or Bregs.

An additional experiment was designed and performed to evaluate CD4+ T‐cell phenotypes. The mouse spleen was acquired under sterile condition, cut into small pieces and filtered with 70‐μm strainer to remove the capsule and connective tissue. The cell supernatant was collected and centrifuged at 1500 rpm for 10 min., with thrice washes atTris‐NH4 cl. FACSAria II flow cytometry (BD Biosciences, San Diego, CA, USA) was used to test spleen CD4+ T‐cell subgroups in animals mentioned above. The CD4+ T cells were isolated by flow cytometry and then were labelled with IFN‐γ‐PE, IL‐4‐PE and Foxp3‐PE antibodies (BD). The proportion of CD4 + IFN‐γ+ T cells, CD4 + IL‐4+ T cells and CD4 + Foxp3+ T cells was accounted. The mRNA expression of T‐bet, GATA‐3 and Foxp3 in lung tissues harvested from various groups was measured on basis of gene probes as listed in Table 1, using Rotor‐Gene 3000 fluorescence ration PCR instrument (Corbett Research, Sydney, Australia).

CTLs were isolated from normal spleen (Mouse CD8 Cells Kit, Invitrogen cat. number 11417D) and pre-incubated with or without recombinant Gal1 (25 μg/ml) for 15 min as described.^{23 (link)} For co-immunoprecipitation, 500 μg cell lysates were incubated with 2 μg anti-FasL or isotype control antibodies (FasL sc-956; isotype control sc-2027; Santa Cruz Biotechnology, Dallas, TX, USA). The immunocomplexes were captured with Protein G PLUS-Agarose (Santa Cruz Biotechnology) and processed for immunoblotting. Cell surface expression of FasL was analyzed in non-permeabilized cells (2 × 10⁵ cells) by flow cytometry. Briefly, purified CD8⁺ T cells were incubated or not with anti-CD3/anti-CD28 mAb (1 μg/μl clone 145-2C11; 1 μg/μl clone 37.51) for 18 h and then stimulated with PBS or Gal1 for the indicated time periods. Cells were analyzed for FasL expression using the anti-FasL mAb (clone MFL3; eBioscience, San Diego, CA, USA). Nonspecific binding determined with isotype-matched control antibodies is shown. Cells were then analyzed on a FACSAria II flow cytometry (BD Biosciences).

ECFC were isolated as previously published.^{18 (link)} Briefly, primary PAEC were sorted by FACS using a FACSAria II flow cytometry cell sorter (BD Biosciences, San Jose, CA, USA) to place a single cell in each well of a collagen-coated 96-well tissue culture plate. Position A1 of each plate received 100 cells as a positive control and position H12 was the negative control. Six plates were prepared from each case, a total of 564 individual cells. These cells, designated clone passage 0 (CP0), were maintained in EGM-2 supplemented with 10% fetal bovine serum (FBS) and examined for the formation of ECFC colonies by light microscopy after 14 days. For the purposes of our experiments, only colonies that covered at least one-eighth of the well were scored. Colonies covering at least one-third of the surface of a 96-well plate were expanded for further analysis, as described below. Colonies that were not grown further were harvested for DNA analysis by proteinase K digestion at 37℃ for 30 min, followed by heat inactivation for 20 min at 95℃, and 2 µL of the crude lysate was used for polymerase chain reaction (PCR).