2.4. Quantification of Total Peripheral CD34+ Positive Cells by Flow Cytometry: CD34+ Is a Marker of Hematopoietic Stem Cells (HSC) Mobilization in Patients

Whole blood samples were used for immnunophenotyping and the total number of peripheral CD34+ cells were quantified by flow cytometry in blood (BD FACSCalibur). The peripheral CD34+ counts were measured following a modified ISHAGE protocol for umbilical cord stem cells detection by flow cytometry (Stem Cell S.A Laboratory, Valladolid, Spain).

Lymphocytoid CD34+ stem cells express low levels of expression of the protein tyrosine phosphatase receptor type C enzyme CD45 (CD45dim). The changes in CD45dim CD34+ stem cell numbers could evaluate the effect of consumption of these nutraceuticals on peripheral CD34 levels in the circulation. The BD Stem reagent (CD34+/CD45+) provided in phosphate-buffered saline (PBS) contains bovine serum albumin (BSA) and 0.1% sodium azide. The reagent contains CD45 FITC, clone 2D1, and CD34 PE, clone 8G12. The kit also contains 7-AAD reagent, which is a nucleic-acid dye used to identify dead cells and 10× ammonium chloride lysing solution. This ammonium chloride solution is a fixative-free solution for red blood cell lysis. The kit contains 50 BD trucount tubes and each single tube contains a freeze-dried pellet of fluorescent beads.

The CD45 recognizes a 180 to 220 Kilodalton (kDa) human leucocyte antigen, which is a member of the leucocyte common antigen (LCA) family. The CD45 antigen is present on all human leucocytes and is weakly expressed on hematopoietic progenitor cells. BD Trucount tubes are used with this reagent. By adding the reagent and the sample directly to the BD Trucount tube, the absolute count of the cell population of interest can be directly determined (BD FACSCalibur^TM flow cytometer). The follows concentrations were used for antibodies.

BD Trucount tubes are used with this reagent by adding the reagent and the sample directly to the BD Trucount tube; the absolute count of the cell population of interest can be directly determined (BD FACSCalibur^TM flow cytometer). The selected volume for samples was 20 μL CD34/CD45, 200 μL as well as 100 μL of blood and 2000 mL of lisis buffer were used for Flow cytometry analysis (see Table 1).

Concentration of antibodies for flow cytometry.