Analysis of murine lymphocyte subpopulations by dual-color flow cytometry: Technical considerations and specificities of monoclonal antibodies directed against surface markers

Platelets may become activated in a number of clinical disorders and participate in thrombus formation. We have developed a direct test for activated platelets in whole blood that utilizes dual-color flow cytometry and requires no washing steps. Platelets were distinguished from erythrocytes and white blood cells in the flow cytometer by labeling the platelets with biotin-AP1, an antibody specific for membrane glycoprotein lb, and analyzing the cells for phycoerythrin-streptavidin fluorescence. Membrane surface changes resulting from platelet activation were detected with three different FITC-labeled monoclonal antibodies: 1) PAC1, an antibody specific for the fibrinogen receptor on activated platelets; 2) 9F9, which binds to the D-domain of fibrinogen and detects platelet-bound fibrinogen; and 3) S12, which binds to an alpha-granule membrane protein that associates with the platelet surface during secretion. Unstimulated platelets demonstrated no PAC1, 9F9, or S12-specific fluorescence, indicating that they did not bind these antibodies. Upon stimulation with agonists, however, the platelets demonstrated a dose-dependent increase in FITC-fluorescence. The binding of 9F9 to activated platelets required fibrinogen. Low concentrations of ADP and epinephrine, which induce fibrinogen receptors but little secretion, stimulated near-maximal PAC1 or 9F9 binding but little S12 binding. On the other hand, a concentration of phorbol myristate acetate that evokes full platelet aggregation and secretion induced maximal binding of all three antibodies. When blood samples containing activated and non-activated platelets were mixed, as few as 0.8% activated platelets could be detected by this technique. There was a direct correlation between ADP-induced FITC-PAC1 binding and binding determined in a conventional 125I-PAC1 binding assay (r = 0.99; p < 0.001). These studies demonstrate that activated platelets can be reliably detected in whole blood using activation-dependent monoclonal antibodies and flow cytometry. This method may be useful to assess the degree of platelet activation and the efficacy platelet inhibitor therapy in thrombotic disorders.

Download Full-text

Determination of freeze-thaw susceptibility of human T-lymphocyte subpopulations using monoclonal antibodies and flow cytometry

Cryobiology ◽

10.1016/0011-2240(81)90172-3 ◽

1981 ◽

Vol 18 (6) ◽

pp. 623

Author(s):

D.M. Strong ◽

J. Budd ◽

T. Williams ◽

F. LaSane ◽

P. DeSantis ◽

...

Keyword(s):

Flow Cytometry ◽

Monoclonal Antibodies ◽

T Lymphocyte ◽

Lymphocyte Subpopulations ◽

Freeze Thaw ◽

T Lymphocyte Subpopulations ◽

Human T Lymphocyte

Download Full-text

Aptamer-Fluorescent Silica Nanoparticles Bioconjugates Based Dual-Color Flow Cytometry for Specific Detection of Staphylococcus aureus

Journal of Biomedical Nanotechnology ◽

10.1166/jbn.2014.1828 ◽

2014 ◽

Vol 10 (7) ◽

pp. 1359-1368 ◽

Cited By ~ 20

Author(s):

Xiaoxiao He ◽

Yuhong Li ◽

Dinggen He ◽

Kemin Wang ◽

Jingfang Shangguan ◽

...

Keyword(s):

Flow Cytometry ◽

Silica Nanoparticles ◽

Specific Detection ◽

Color Flow ◽

Fluorescent Silica Nanoparticles ◽

Dual Color

Download Full-text

Mono-Chlorinated 3-Carboxy-7-Hydroxycoumarin: A Highly Fluorescent and Water-Soluble Violet Excitable Dye for Cell Analysis

Blood ◽

10.1182/blood.v112.11.4929.4929 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4929-4929

Author(s):

Barny Abrams

Keyword(s):

Flow Cytometry ◽

Quantum Yield ◽

Adverse Effects ◽

Water Soluble ◽

Surface Markers ◽

Cell Surface Markers ◽

Cell Analysis ◽

Color Flow ◽

Antibody Conjugates ◽

Photochemical Properties

Abstract In our search for new violet excitable dyes with improved photochemical properties, we examined several halogen substituted 7-hydroxycoumarins and found that chlorinated derivatives could be as bright as their fluorinated analogs. In particular, mono-chlorinated 3-carboxy-7-hydroxycoumarin (V450) has a quantum yield equal to 3-carboxy-6,8- difluoro-7-hydroxycoumarin (Pacific Blue™). Antibodies specific to various cell surface markers (CD3, CD4, CD45) were labeled with V450 and tested in single color flow cytometry assays. Our results show that conjugates of V450 are brighter than the same clones labeled with Pacific Blue™. Additional studies were performed in which V450-antibody conjugates were included in 8-color cocktails, along with FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, and APC-H7 conjugates, aimed at subsetting lymphocytes and myeloid cells. These cocktails met all standards of performance for the particular applications for which they were designed, including stability, lack of interaction between components, and brightness. The new V450 fluorochrome proved to be compatible with both FACSLyse and PharmLyse processing of EDTA anti-coagulated whole blood, and showed no signs of adverse effects on unfixed cells.

Download Full-text