For the assay of flow cytometry, 1-2×105 cells were seeded per well in 6-well plates containing complete medium. After cell adhesion, 10 μg FITC-UEA (Sigma) or Fluorescein labeled LCA (Vector lab) and appropriate amount of PBS were added to each well until the total volume was 1000 μl. After being gently mixed, the samples were incubated at 37 °C in darkness for 1 h. Then, the cells were detached with 0.2% trypsin in darkness and the re-suspended single-cell suspensions were fixed with 1% paraformaldehyde for 20 min. The fluorescence intensity was analyzed using a FACS Calibur machine (BD).
Fitc uea
Manufactured by Merck Group
Sourced in United States
FITC-UEA is a fluorescently labeled lectin derived from the seeds of the Japanese pagoda tree (Sophora japonica). It specifically binds to terminal α-L-fucose residues on glycoproteins and glycolipids, and is commonly used as a histological and cytological marker for the detection of these residues in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde (pfa), by Merck Group (3 mentions)
Dii ac ldl, by Thermo Fisher Scientific (2 mentions)
Fluorescein labeled lca, by Vector Laboratories (1 mentions)
Facscalibur machine, by BD (1 mentions)
Fluorescein isothiocyanate (fitc), by Santa Cruz Biotechnology (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions)
Amd3100, by Merck Group (1 mentions)
L methionine, by Merck Group (1 mentions)
Ficoll, by Merck Group (1 mentions)
Su5416, by Merck Group (1 mentions)
Phosphor p38 mapk, by Cell Signaling Technology (1 mentions)
Sdf 1, by Cell Signaling Technology (1 mentions)
P38 mapk, by Cell Signaling Technology (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Gapdh antibody, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
6 protocols using fitc uea
1
Flow Cytometry Lectin Binding Assay
2
Endothelial Cell Identification
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After incubation with DiI-ac-LDL, the selected cell population was washed twice with PBS and incubated with 5 μg/ml FITC-UEA (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) at 37°C and 5% CO2. After an additional washing step with PBS, cells were analyzed by fluorescence microscopy.
3
Characterization of Endothelial Progenitor Cells
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After being cultured for 10 days, MNCs were incubated with l,l’-dioctadecyl-1,3,3,3’,3’-tetramethyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-ac-LDL, Peking Union-Biololgy Co. Ltd, Beijing, China, catalog number: N/A) (2.5 mg/mL) for 2 h at 37 °C, and then fixed with 2% paraformaldehyde (Sigma, catalog number: P6148,) for 5 min. Thereafter, the cells were washed with DPBS (ThermoFisher, Waltham, MA, USA, catalog number: 14190250) for three times and incubated with FITC-UEA (10 mg/L, Sigma, catalog number: L9006) for 1 h at 37 °C.
After being fixed in 2% paraformaldehyde (Sigma, catalog number: P6148) for 10 min, the cells were incubated with primary antibodies against CD133 (Abcam, Cambridge, UK, catalog number: ab16518) and FLK-1 (Abcam, catalog number: ab9530) for 1 h at 37 °C. After being washed with PBS for three times, EPCs were incubated with secondary antibodies conjugated with Cy3 (BOSTER, catalog number: BA1031, Pleasanton, CA, USA) or FITC (Santa Cruz, DBA, Milan, Italy, catalog number: SC-2359) for 30 min at 37 °C. Then a representative micrograph was acquired by a fluorescence microscope (Olympus, Tokyo, Japan).
After being fixed in 2% paraformaldehyde (Sigma, catalog number: P6148) for 10 min, the cells were incubated with primary antibodies against CD133 (Abcam, Cambridge, UK, catalog number: ab16518) and FLK-1 (Abcam, catalog number: ab9530) for 1 h at 37 °C. After being washed with PBS for three times, EPCs were incubated with secondary antibodies conjugated with Cy3 (BOSTER, catalog number: BA1031, Pleasanton, CA, USA) or FITC (Santa Cruz, DBA, Milan, Italy, catalog number: SC-2359) for 30 min at 37 °C. Then a representative micrograph was acquired by a fluorescence microscope (Olympus, Tokyo, Japan).
4
Characterizing MNCs from Mice Bone Marrow
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MNCs from mice bone marrow were cultured for 10 days, incubated with 2.5 mg/L DiI-ac-LDL (Thermo Fisher Scientific Inc., Waltham, MA, USA) for 2 h at 37 °C, and 10 mg/L FITC-UEA (Sigma-Aldrich, St Louis, MO, USA) for 1 h at 37 °C, and then fixed for 5 min with 1–2% paraformaldehyde (Sigma-Aldrich, St Louis, MO, USA). Double-positive cells and total cell numbers were counted in five random fields under a fluorescence microscope (× 100). The positive cells (%) represent the numbers of double-positive cells compared with the total number of cells.
5
Ginsenoside Rb1 Enhances Stem Cell Therapy
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Ginsenoside Rb1(Rb1) purchased as a reference compound (purity > 98%) from the Division of Chinese Materia Medica and Natural Products, National Institute for the Control of Pharmaceutical and Biological Products (NICPBP), Ministry of Public Health, China. EGM-2MV was purchased from Lonza (Switzerland). DiI-ac-LDL was purchased from Molecular Probe (Thermo Scientific, Waltham, MA, USA). Antibodies against Flk-1, CD133 and CD34 were purchased from Bioss (Beijing, China). Trypsin, penicillin, streptomycin, CM-DiI and FBS were purchased from Gibco (Invitrogen, Carlsbad, CA, USA). Fibronetin was purchased from BD Biosciences (San Jose, CA, USA). L-methionine, SST, FITC-UEA, Hcy, Ficoll, H2DCF-DA, DAPI, Evans blue, AMD3100, SU5416 and GAPDH antibody were purchased from Sigma (St Louis, MO, USA). SDF-1 ELISA Kit was purchased from Cusabio (Wuhan, China). Antibodies against p38MAPK, phosphor-p38MAPK, SDF-1, VEGFR2 and tubulin were purchased from Cell Signaling Technology (Boston, MASS, USA). Fogarty catheter was purchased from Edwards Lifesciences (Irvine, CA, USA).
6
Dual-Labeled Cell Characterization
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After cultured for 7 days, cells seeded in 24-well plates were incubated with 2.5 mg/L DiI-ac-LDL (Thermo Fisher Scienti c Inc.) for 2 h at 37°C and 10mg/L FITC-UEA (Sigma-Aldrich) for 1 h at 37°C, and then xed for 5 min with 1-2% paraformaldehyde (Sigma-Aldrich). Double positive cells and total cell numbers were counted in ve random elds.
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