Fluoro‐Gold: An alternative viability stain for multicolor flow cytometric analysis

Abstract

Background:The viability stains propidium iodide (PI) and 7-amino-actinomycin D (7-AAD) are excited at 488 nm, as are the commonly used antibody conjugates fluorescein isothiocyanate (FITC), phycoerythrin (PE), and cyanine 5 dye covalently coupled to R-phycoerythrin (RPE-Cy5). When excited by a single laser, spectral overlap in the emission of PI and 7-AAD with RPE-Cy5 precludes the use of these viability stains for three-color immunophenotyping, particularly when evaluating low levels of marker expression in viable target cells. The ultraviolet excitable dye hydroxystilbamidine methanesulfonate (Fluoro-Gold, or FG) binds to DNA at the A-T-rich regions of the minor groove in permeabilized or dead cells. We assessed the suitability of this dye as a viability stain. Methods: The ability of FG to detect nonviable cells in fresh and cryopreserved human apheresed peripheral blood cells was compared with that of PI and 7-AAD. The stability of FG staining and the effects of dye and cell

Endogenous Fibroblastic Progenitor Cells in the Adult Mouse Lung Are Highly Enriched in the Sca-1 Positive Cell Fraction

“…FcγIII/II receptor block was performed with anti‐mouse CD16/32 followed by staining with directly conjugated rat anti‐mouse CD45, CD31, Ter119, Sca‐1, and appropriate isotype controls (BD Biosciences). The fluorochromes, 7‐amino‐actinomycin D (2 μg/ml; Sigma) or Fluoro‐gold (FG) [16] (1 μg/ml; Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com) were used as viability dyes for flow cytometric detection and exclusion of nonviable cells.…”

Guidelines on the use of multicolour flow cytometry in the diagnosis of haematological neoplasms

“…The use of nuclear dyes may be helpful for analysis where more exact determination of neoplastic cells is required, such as MRD analysis; and for establishing/confirming the presence of nucleated erythroid cells. Several reagents are available; examples of reports on their relative merits in the context of multi‐colour panels include Barber et al (), Perfetto et al () and Mahnke and Roederer ().…”

Myeloma plasma cells alter the bone marrow microenvironment by stimulating the proliferation of mesenchymal stromal cells

“…Lineage-negative (Lin -) cells were subsequently analyzed based on the expression of CD45, CD31, CD51 and Sca-1 phenotypic markers and viable cells were gated by fluorogold exclusion. 35 Initially, the viable cell populations were sorted and the GFP Figure 6A-D). At 28-days post tumor inoculation, tumor-bearing mice exhibited a significant increase (P<0.05, t-test) in the proportion of MSC within the bone and significant decrease (P<0.05, t-test) in OB numbers compared with controls ( Figure 6E and F).…”