Nonspecific binding of conjugated antibodies represents a critical step which could significantly influence the results of immunostaining or flow cytometry. In this respect, various staining procedures and distinct cell types can alter the results obtained with different fluorochromes. In this study, we analysed nonspecific binding of R-phycoerythrin (R-PE)-conjugated antibodies to mouse mitogen-stimulated B and T lymphocytes. The cells were fixed, permeabilized and stained using isotype control antibodies conjugated with different fluorochromes and assessed by flow cytometry. R-PE-conjugated antibodies bound to LPS-stimulated B cells, in contrast to Con A-stimulated T cells, independently of their specificity. The percentage of R-PE positive B cells varied, according to the used antibodies or the fixation/permeabilization kit. Nevertheless, up to 30% of R-PE+ B cells after staining with R-PE-conjugated isotype control antibodies was detected. Furthermore, LPS-stimulated B cells bound nonspecifically, in a dose-dependent manner, unconjugated R-PE molecules. Con A-stimulated T cells slightly bound R-PE only in high concentrations. Similarly, the antibodies conjugated with other fluorochromes showed less than 1% of nonspecific binding independently of the manufacturer of antibodies or fixation/permeabilization kits. The data demonstrated that LPS-stimulated B cells, in contrast to Con A-stimulated T cells, bind R-PE nonspecifically following formaldehyde or paraformaldehyde fixation. Therefore, the results based on the use of R-PE-conjugated antibodies should be taken with a precaution.

• MeSH
• B-Lymphocytes immunology   MeSH
• Phycoerythrin immunology   metabolism   MeSH
• Mitogens immunology   MeSH
• Antibodies, Monoclonal immunology   MeSH
• Mice, Inbred BALB C MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• T-Lymphocytes immunology   MeSH
• Binding Sites immunology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The Black Sea is the largest meromictic sea with a reservoir of anoxic water extending from 100 to 1000 m depth. These deeper layers are characterised by a poorly understood fluorescence signal called "deep red fluorescence", a chlorophyll a- (Chl a) like signal found in deep dark oceanic waters. In two cruises, we repeatedly found up to 103 cells ml-1 of picocyanobacteria at 750 m depth in these waters and isolated two phycoerythrin-rich Synechococcus sp. strains (BS55D and BS56D). Tests on BS56D revealed its high adaptability, involving the accumulation of Chl a in anoxic/dark conditions and its capacity to photosynthesise when re-exposed to light. Whole-genome sequencing of the two strains showed the presence of genes that confirms the putative ability of our strains to survive in harsh mesopelagic environments. This discovery provides new evidence to support early speculations associating the "deep red fluorescence" signal to viable picocyanobacteria populations in the deep oxygen-depleted oceans, suggesting a reconsideration of the ecological role of a viable stock of Synechococcus in dark deep waters.

• MeSH
• Chlorophyll A metabolism   MeSH
• Ecosystem MeSH
• Fluorescence MeSH
• Photosynthesis MeSH
• Phycoerythrin metabolism   MeSH
• Phylogeny MeSH
• Genome, Bacterial MeSH
• Oceans and Seas MeSH
• Synechococcus chemistry   classification   isolation & purification   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Black Sea MeSH
• Oceans and Seas MeSH

Even though superresolution microscopy indicates that size of plasma membrane rafts is <20 nm, those structures have never been observed. Förster resonance energy transfer (FRET) is therefore still the most powerful optical method for characterization of such domains. In this letter we investigate relation between nanodomain affinity of a donor-acceptor (D/A) pair and the detectable nanodomain size/area. We show that probes with high affinity to the liquid-ordered (L(o)) phase are required for detecting domain sizes of a few nanometers, and/or domains that occupy a few percent of the bilayer area. A combination of donors and acceptors that prefer different phases is the more favorable approach. For instance, a D/A pair with the distribution constant of donors K(D) = 5 and acceptors K(A) = 0.01 can resolve a broad spectrum of nanodomain sizes. On the other hand, currently available donors and acceptors that prefer the same phase, either the liquid-disordered (L(d)) or L(o) phase, are not so convenient for determining domain sizes <20 nm. Here the detection limits of FRET experiments employing several commonly used D/A pairs have been investigated.

• MeSH
• Time Factors MeSH
• Cholera Toxin chemistry   MeSH
• Electrons MeSH
• Phycoerythrin chemistry   MeSH
• Carbocyanines chemistry   MeSH
• Lipid Bilayers chemistry   MeSH
• Membrane Microdomains chemistry   MeSH
• Monte Carlo Method MeSH
• Nanoparticles chemistry   MeSH
• Perylene chemistry   MeSH
• Reproducibility of Results MeSH
• Fluorescence Resonance Energy Transfer methods   MeSH
• Rhodamines chemistry   MeSH
• Boron Compounds chemistry   MeSH
• Particle Size MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

In the present study, we compared three single platform methods for CD34+ hematopoietic stem cell (HSC) enumeration by flow cytometry. For this purpose, we analyzed the performance characteristics and results obtained from different HSC sources. Interlaboratory coefficients of variation (CV) for precision/reproducibility analysis varied from 4.0% to 6.7% / 6.7% to 9.2% for the low and 3.2% to 4.1% / 4.3% to 6.7%, respectively, for the high stem cell control. Correlation between methods ranged from 0.92% to 0.99%; Wilcoxon test showed no significant differences (p > 0.05); Bland-Altman analysis confirmed good agreement between assays (mean bias ranging from -0.48 to 6.91). Our results demonstrate very good intralaboratory correlation and agreement between methods, confirm the major impact of single platform strategy for accurate and reproducible HSC enumeration and suggest that high interlaboratory variability could be influenced by incorrect performance of validated methods.

• MeSH
• Antigens, CD34 analysis   immunology   MeSH
• Bone Marrow Cells MeSH
• Dactinomycin analogs & derivatives   MeSH
• Granulocyte Colony-Stimulating Factor pharmacology   MeSH
• Fluorescein-5-isothiocyanate MeSH
• Fluorescent Dyes MeSH
• Phycoerythrin MeSH
• Hematopoietic Stem Cells MeSH
• Blood Cells MeSH
• Blood Cell Count MeSH
• Laboratories MeSH
• Leukapheresis MeSH
• Humans MeSH
• Hematopoietic Stem Cell Mobilization MeSH
• Antibodies, Monoclonal immunology   MeSH
• Cell Count methods   MeSH
• Antineoplastic Agents pharmacology   MeSH
• Flow Cytometry methods   MeSH
• Reproducibility of Results MeSH
• Sensitivity and Specificity MeSH
• Antibody Specificity MeSH
• Bone Marrow Examination MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Comparative Study MeSH

• MeSH
• Biliverdine analogs & derivatives   MeSH
• Eukaryota genetics   MeSH
• Photosynthesis MeSH
• Phycocyanin biosynthesis   MeSH
• Phycoerythrin biosynthesis   MeSH
• Proteins MeSH
• Publication type
• Review MeSH