Pe conjugated cd14

Cultured cells (passage 1) were trypsinized and stained with a panel of antibodies for fluorescence-activated cell sorting (FACS) analysis. Approximately, 1 × 10⁵ cells were re-suspended in phosphate buffered saline (PBS) and incubated with IgG block for 5 minutes to block non-specific binding. The following antibodies were used: AF-700 conjugated CD3 (BD BioSciences, USA), PE conjugated CD14 (BD, Immunocytometry, USA), APC conjugated CD19 (BD BioSciences, USA), PE conjugated CD34 (BD, BioSciences, USA), APC conjugated CD44 (BD, Pharmingen, USA), FITC conjugated CD45 (BD Pharmingen, USA), PE conjugated CD73 (BD Pharmingen, USA), AF-700 conjugated CD90 (Biolegend, USA) and APC conjugated CD105 (Biolegend, USA). Cells were stained for 30 minutes at 4°C with the antibodies. After washing, samples were analyzed on a LSR II flow cytometer (BD, USA) and at least 10,000 events were acquired for each population. Data acquisition and analysis were performed using FACS DIVA software (BD Biosciences, USA). Unstained cells were used to establish flow cytometer settings. Debris and cells/particles with auto-fluorescence were removed by using a threshold on the forward scatter.

For the CD90+ MSC authentication, after isolation of the mononuclear cells, we set apart a 5 × 10⁵ cell aliquot in 1 mL for evaluating the presence of stem cell markers by means of FC. The marking procedure was as follows: once the cells had been separated from the Ficoll and washed with PBS, a portion of approximately 2.5 × 10⁴ cells are placed in polystyrene tubes [Falcon; Becton–Dickinson (BD)] with 10 µL of the antibody suspension and were left to incubate for 30 min at 4 °C. The monoclonal (directly conjugated) antibodies applied were: FITC-conjugated CD90 (50 μg/ml, mouse IgG1κ, cat. 555595), PE-conjugated CD14 (20 μg/ml, mouse IgG2bκ, cat. 340660), FITC-conjugated CD105 (5 μg/ml, mouse IgG1κ, cat. 561443), and PE-conjugated CD166 (20 μg/ml, mouse IgG1κ, cat. 559263) all from BD PharMigen™ (California, USA). The samples and or unlabelled controls were included for each antibody and used to set the gating on the flow cytometer. Data were acquired in a BD FACSCalibur flow cytometer and analyzed by CellQuest™ PRO software (Becton–Dickinson) with a mean of 20,000 events. This procedure was repeated each time that CD90+ cells were obtained from the sheep.

To evaluate surface markers associated with fibroblasts and macrophage, a flow cytometry (FC) assay was performed according to Landa-Solís C et al. [16 (link)] Cells were marked with monoclonal antibodies PE-conjugated CD166 and PE-conjugated CD14 from BD PharMigenTM (San Diego, CA, USA). Data were collected through a BD FACSCalibur flow cytometer and analyzed with CellQuestTM PRO software (Becton-Dickinson).