Pe conjugated cd133

Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll gradient centrifugation (Amersham Biosciences, Freiburg, Germany). Cell-surface antigens expression was quantified by Fluorescence Activated Cell Sorting (FACS) analysis as described previously [8 (link)]. The following anti-human monoclonal antibodies have been used: PE-conjugated CD133 (Miltenyi Biotec, Bergisch-Gladbach, Germany), PerCP-conjugated CD34 (BD Biosciences, Heidelberg, Germany), and either FITC-conjugated VCAM-1/CD106, FITC-conjugated ICAM-1/CD54, FITC-conjugated E-selectin/CD62E and FITC-conjugated L-selectin/CD62L. Flow cytometry was executed on a FACSCalibur flow cytometer (BD Biosciences) and data analysis was performed using WinMDI 2·8 software (Scripps Research Institute, La Jolla, CA). CD34⁺/CD133⁺-stem cell counts are expressed as percentage of total PBMC in each patient or control.

Cells were dissociated using Accutase and resuspended in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA). The cells were stained with PE-conjugated CD133 (130-098-826; Miltenyi Biotec) or isotype control antibody (BD555742; BD Biosciences) on ice for 30 min. Cells were then washed with PBS and analyzed on a BD FACSCalibur^™ (BD Biosciences) using BD CellQuest software (BD Biosciences).

Cells were fixed on ice for 20 min with 2% paraformaldehyde, blocked with 10% NGS for 1 h, followed by primary antibody incubation with PE-conjugated CD133 (Miltenyi) for 30 min. Labeled cells were analyzed using a BD Calibur, with unlabeled cells serving as negative controls. For cell cycle analysis, cells were pre-treated for 24 h with 100 ng/mL Sema3A, dissociated, and fixed with cold 70% ethanol for 30 min at 4 ^oC. Cells were washed and treated with RNAse (Qiagen). Propidium Iodide (PI) was added to the cells and analysis was conducted using a BD Calibur, with post-analysis using FlowJo software version 7.6.5.

FACS analysis and cell sorting were performed using FACS Calibur and FACS Aria machines (Becton Dickinson, Palo Alto, CA), respectively. FACS data were analyzed using Flowjo software (Tree Star, Ashland, OR). Antibodies to the following proteins were used: PE-conjugated CD133 (dilution 1/40, MACS; Miltenyi Biotech, Sunnyvale, CA, Germany, 130-080-801) and PE-conjugated ALDH1 (dilution 1/40, StemCell Technologies, Durham, NC, USA, 01700). The FACS gates were established by staining with isotype antibody or secondary antibody.

The antibodies used included PerCP-Cy5.5-conjugated CD44 (eBioscience, USA), PE-Cy7-conjugated EpCAM (eBioscience, USA), APC-conjugated CD90 (eBioscience, USA) and PE-conjugated CD133 (Miltenyi Biotec, Germany). Isotype-matched immunoglobulins were served as controls.

FACS analysis and cell sorting were performed using FACS Calibur and FACS Aria machines (Becton Dickinson, Palo Alto, CA), respectively. FACS data were analyzed using FlowJo software (Tree Star, Ashland, OR). Antibodies to the following proteins were used: APC-conjugated CD44 (BD Bioscience, Cat. 559942, dilution 1/40), PE-conjugated CD133 (MACS; Miltenyi Biotech, Sunnyvale, CA, 130-080-081, dilution 1/40), CD34 (MACS; Miltenyi Biotech, Sunnyvale, CA, 30-081-002,dilution 1/40), CD44 (MACS; Miltenyi Biotech, Sunnyvale, CA, 130-095-180, dilution 1/40), CD45 (MACS; Miltenyi Biotech, Sunnyvale, CA, 130-080-201, dilution 1/40), CD73 (MACS; Miltenyi Biotech, Sunnyvale, CA, 130-095-182, dilution 1/40), CD105 (MACS; Miltenyi Biotech, Sunnyvale, CA, 130-094-941, dilution 1/40), Annexin V (BD Bioscience, Cat. No. 556547, dilution 1/40), and propidium iodide (PI) (Invitrogen, Cat No P3566, dilution 1/40). Add 5 μl of antibody from each dilution into separate sample tubes containing cells (4 × 10⁵). Mix well and incubate cells on ice for 30 min. Wash with 10 ml of medium. The FACS gates were established by staining with an isotype antibody.