The expression of cell-surface antigens was quantified by immunostaining as described previously [7 (link)]. We used the following monoclonal antibodies (anti-human): PE-conjugated CD133 (Miltenyi Biotec, Bergisch-Gladbach, Germany), PerCP-conjugated CD34 (BD Biosciences, Heidelberg, Germany), and either FITC-conjugated CXCR-4 or APC-conjugated c-Kit, APC-conjugated CXCR-2 (all R&D Systems, Wiesbaden-Nordenstadt, Germany), PE-conjugated PSGL1 (BD Biosciences, Heidelberg, Germany) or the indirect rabbit anti-human polyclonal RAGE antibody (Biozol, Eching, Germany), for which a FITC-conjugated anti-rabbit IgG antibody (Invitrogen, Karlsruhe, Germany) was used. We used a FACSCalibur flow cytometer (BD Biosciences) for flow cytometry. FACS-data analysis was performed with WinMDI 2.8 software (Scripps Research Institute, La Jolla, CA). EPC counts are expressed as percentage referred to total PMBC in each study subject.
Percp conjugated cd34
Manufactured by BD
Sourced in Germany
PerCP-conjugated CD34 is a fluorescently labeled antibody that binds to the CD34 antigen. CD34 is a cell surface glycoprotein expressed on hematopoietic stem and progenitor cells. The PerCP fluorescent dye is used to label the CD34 antibody, allowing for the detection and identification of CD34-positive cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Pe conjugated cd133, by Miltenyi Biotec (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Fitc conjugated anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
Apc conjugated cd45, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Ficoll gradient centrifugation, by Cytiva (1 mentions)
4 protocols using percp conjugated cd34
1
Quantifying Cell-Surface Antigens by Flow Cytometry
2
Identification of Endothelial Progenitor and Circulating Endothelial Cells
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Blood samples were collected from freshly placed venous cannulas. Fifty microliters of
blood sample were incubated with 5 µL of FITC-labeled CD144 (BD Biosciences),
PE-conjugated CD133 (AC133; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany),
Per-CP-conjugated CD34 (BD Biosciences), and APC-conjugated CD45 (BD Biosciences) for 20
minutes. After incubation, RBC lysis and washing were done and the cells were suspended
in PBS, and FACSCalibur flow cytometric analysis was done with Cell Quest software (BD
Biosciences). Antihuman IgG was used as an isotype negative control and 50 000 events
were analyzed. The EPCs are negative for CD45, positive for CD144, CD34, and CD133
(CD45– CD34+ CD144+ CD133+), while CECs
are negative for CD45, positive for CD144 and CD34 and negative for CD133
(CD45– CD34+ CD144+ CD133–). The CECs and
EPCs were expressed as absolute count per 50 000 cells (Figure 2 ).
blood sample were incubated with 5 µL of FITC-labeled CD144 (BD Biosciences),
PE-conjugated CD133 (AC133; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany),
Per-CP-conjugated CD34 (BD Biosciences), and APC-conjugated CD45 (BD Biosciences) for 20
minutes. After incubation, RBC lysis and washing were done and the cells were suspended
in PBS, and FACSCalibur flow cytometric analysis was done with Cell Quest software (BD
Biosciences). Antihuman IgG was used as an isotype negative control and 50 000 events
were analyzed. The EPCs are negative for CD45, positive for CD144, CD34, and CD133
(CD45– CD34+ CD144+ CD133+), while CECs
are negative for CD45, positive for CD144 and CD34 and negative for CD133
(CD45– CD34+ CD144+ CD133–). The CECs and
EPCs were expressed as absolute count per 50 000 cells (
3
Quantification of Circulating and Endothelial Progenitor Cells
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Whole blood samples from patients were processed within 2 h of collection. Samples were assessed by flow cytometry on an LSR II (BD Biosciences, San Jose, CA) using the following antibodies: Allophycocyanin-conjugated CD133 (Miltenyi Biotec, Auburn, CA), PerCP-conjugated CD34 (BD Biosciences), and PE-conjugated VEGFR2 (R&D Systems, Emeryville, CA). Data were analyzed with FlowJo software (Tree Star, Ashland, OR). Patient samples were performed in triplicate; the mean of three runs was used as CPC and EPC levels. To assess interassay variability, five samples from one patient were drawn on five separate days and analyzed with consistent results (<5% variation). CPCs were quantified as cells positive for CD34 and CD133 markers. EPCs were quantified as cells positive for CD34, CD133, and VEGFR2 markers.
4
FACS Analysis of PBMC Stem Cells
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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.
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