Cells were washed and counted with a hemocytometer. Mononuclear cells were immunostained with monoclonal anti-bodies against human CD34+ for each group of analyses, one set of control tubes for machine calibration was generated. Flow cytometry was performed in a laboratory. The forward side scatter plot was used to identify lymphocyte gate. 100.000 CP-690550 events per sample were acquired. Total cell count was averaged. The principle, clinical applications precautions and methodology are as follows: IOTest CD34+ PE The use of this fluorochrome-conjugated antibody permits the identification and numeration of cell populations expressing the CD34+ antigen present in human biological samples using flow cytometry. Principle This test is based on the ability of specific monoclonal antibodies to bind to the antigenic determinants expressed by leucocytes.
Specific staining of the leucocytes is performed by incubating the sample with the IOTest reagent. The red cells are then removed by lysis and the leucocytes, which are unaffected by this process, and then, analyzed by flow cytometry. The flow cytometer measures light diffusion and the fluorescence of cells. It makes possible the delimitation of the population of interest (cells) within the electronic window defined on a histogram, which correlates the orthogonal diffusion of light (Side Scatter or SS) and the diffusion of narrow angle light (Forward Scatter or FS). Other histograms combining two of the different parameters available on the cytometer can be used as supports in the gating stage depending on the application chosen by the user.
The fluorescence of the delimited cells is analyzed in order to distinguish the positively stained events from the unstained ones. The results are expressed as a percentage of positive events in relation to all the events acquired by the gating. Procedure Note: The procedure below is valid for standard applications. Sample and/or Versa Lyse volumes for certain Beckman Coulter applications may be different for each sample analyzed. In addition to the test tube, one control tube is required in which the cells are mixed in the presence of the otypic control (Ref. A07796). Add 20 ��L of specific IOTest conjugated antibody to each test tube, and 20 ��L of the isotypic control to each control tube. Add 100 ��L of the test sample to both tubes. Vortex the tubes gently.
Incubate for 15 to 20 minutes at room temperature (18 �C 25��C), protected from light. Then perform lysis of the red cells, if necessary, by following the recommendations of the lysis reagent used. As an example, if you wish to use VersaLyse (Ref. A09777), refer to the leaflet and follow preferably the procedure called ��with concomitant fixation��, which consists of adding 1 ml of the ��Fix-and-Lyse�� mixture prepared extemporaneously. Anacetrapib Vortex immediately for one second and incubate for 10 minutes at room temperature, protected from light.