Time-gated flow cytometry: an ultra-high selectivity method to recover ultra-rare-event μ-targets in high-background biosamples

2009 ◽  
Vol 14 (2) ◽  
pp. 024023 ◽  
Author(s):  
Dayong Jin ◽  
James A. Piper ◽  
Robert C. Leif ◽  
Sean Yang ◽  
Belinda C. Ferrari ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Selectivity ◽  
Rare Event ◽  
High Background

UV LED excited time-gated luminescence flow cytometry: evaluation for rare-event particle counting

2008 ◽  
Author(s):  
Dayong Jin ◽  
Belinda Ferrari ◽  
Robert C. Leif ◽  
Sean Yang ◽  
Lidia M. Vallarino ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Rare Event ◽  
Uv Led ◽  
Particle Counting

scholarly journals Setting objective thresholds for rare event detection in flow cytometry

2014 ◽  
Vol 409 ◽  
pp. 54-61 ◽  
Author(s):  
Adam J. Richards ◽  
Janet Staats ◽  
Jennifer Enzor ◽  
Katherine McKinnon ◽  
Jacob Frelinger ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Event Detection ◽  
Rare Event

scholarly journals Use of fluorescence threshold triggering and high-speed flow cytometry for rare event detection

Cytometry ◽  
1995 ◽  
Vol 22 (4) ◽  
pp. 317-322 ◽  
Author(s):  
Mark A. Rehse ◽  
Stan Corpuz ◽  
Shelly Heimfeld ◽  
Mark Minie ◽  
Diane Yachimiak
Keyword(s):  
Flow Cytometry ◽  
Event Detection ◽  
High Speed ◽  
Rare Event ◽  
High Speed Flow ◽  
Speed Flow

The Use of Allele-Specific Recombinant Fcγ Receptor IIIb Antigens for the Detection of Granulocyte Antibodies

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 357-362 ◽  
Author(s):  
Juergen Bux ◽  
Karin Kissel ◽  
Christine Hofmann ◽  
Sentot Santoso
Keyword(s):  
Flow Cytometry ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Fcγ Receptor ◽  
High Background ◽  
Mammalian Expression ◽  
Human Sera ◽  
Allele Specific ◽  
Granulocyte Antibodies

The Fcγ receptor IIIb (FcγRIIIb) for the Fc domain of IgG is expressed exclusively on neutrophils. The FcγRIIIb bears allotypic polymorphisms referred to as NA1, NA2, and SH, which are known for their frequent involvement in alloimmune and autoimmune neutropenias as well as in transfusion reactions. The bactericidal capacity of isolated neutrophils is easily activatable, and activation results in self-desintegration, thus preventing storage of neutrophils. As a result, only freshly isolated granulocytes can be used for antibody screening, often making it impossible to use typed panel cells. To provide a readily available source of typed panel cells, we therefore established stable mammalian cells expressing recombinant NA1, NA2, and SH antigens. We isolated mRNA from typed neutrophils and then transcribed it in cDNA. The cDNA that codes for the different forms of the FcγRIIIb was amplified by polymerase chain reaction and was subsequently subcloned into the mammalian expression vector pcDNA3. Chinese hamster ovary (CHO) cells were transfected with allele-specific constructs, and stable cell lines expressing FcγRIIIb were selected by flow cytometry. Because human sera show high background fluorescence with transfectants in flow cytometry, the monoclonal antibody–specific isolation of granulocyte antigens (MAIGA) assay was performed. By MAIGA assay, we tested 14 well-characterized human alloantibodies directed against the antigens NA1, NA2, and SH; 5 FcγRIIIb-specific isoantibodies; and 12 FcγRIIIb-reactive autoantibodies. Except one NA1- and one SH-specific alloantibody, all other antibodies could be identified by the use of CHO transfectants. In contrast to neutrophils, fixed CHO cells can be stored at 4°C for at least 4 weeks or stored frozen for a longer period. This longer shelf life of the transfected CHO cells compared with isolated neutrophils will simplify the detection of the clinically most important FcγRIIIb-reactive alloantibodies and autoantibodies.

scholarly journals A critical evaluation of a flow cytometer used for detecting enterococci in recreational waters

2007 ◽  
Vol 5 (2) ◽  
pp. 295-305 ◽  
Author(s):  
Dawn N. King ◽  
Kristen P. Brenner ◽  
Mark R. Rodgers
Keyword(s):  
Flow Cytometry ◽  
Environmental Protection Agency ◽  
Detection System ◽  
Membrane Filter ◽  
Critical Evaluation ◽  
Flow Cytometer ◽  
Target Cells ◽  
Recreational Water ◽  
Recreational Waters ◽  
High Background

The current U. S. Environmental Protection Agency-approved method for enterococci (Method 1600) in recreational water is a membrane filter (MF) method that takes 24 hours to obtain results. If the recreational water is not in compliance with the standard, the risk of exposure to enteric pathogens may occur before the water is identified as hazardous. Because flow cytometry combined with specific fluorescent antibodies has the potential to be used as a rapid detection method for microorganisms, this technology was evaluated as a rapid, same-day method to detect enterococci in bathing beach waters. The flow cytometer chosen for this study was a laser microbial detection system designed to detect labeled antibodies. A comparison of MF counts with flow cytometry counts of enterococci in phosphate buffer and sterile-filtered recreational water showed good agreement between the two methods. However, when flow cytometry was used, the counts were several orders of magnitude higher than the MF counts with no correlation to Enterococcus spike concentrations. The unspiked sample controls frequently had higher counts than the samples spiked with enterococci. Particles within the spiked water samples were probably counted as target cells by the flow cytometer because of autofluorescence or non-specific adsorption of antibody and carryover to subsequent samples. For these reasons, this technology may not be suitable for enterococci detection in recreational waters. Improvements in research and instrument design that will eliminate high background and carryover may make this a viable technology in the future.

Rare-Event Analysis in Flow Cytometry

2007 ◽  
Vol 27 (3) ◽  
pp. 627-652 ◽  
Author(s):  
Albert D. Donnenberg ◽  
Vera S. Donnenberg
Keyword(s):  
Flow Cytometry ◽  
Rare Event ◽  
Event Analysis

scholarly journals Desialylation and Apoptosis Crosstalk to Modulate Platelet Clearance

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1055-1055
Author(s):  
Renata Grozovsky ◽  
Cameron Fraser ◽  
Karin M. Hoffmeister ◽  
Kristopher Sarosiek ◽  
Silvia Giannini
Keyword(s):  
Flow Cytometry ◽  
Cytochrome C ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
Small Cells ◽  
Cytochrome C Release ◽  
Apoptotic Pathway ◽  
Platelet Surface ◽  
High Background

Our understanding of cell biological processes involved in aging has advance greatly over the past decades. Platelets are small cells that circulate for 4-5 days in mice and 7-10 days in humans. And even though, platelets are anucleated cells, a growing body of evidence shows that platelet clearance is a well-regulated mechanism. We have recently demonstrated that platelets lose sialic acid as they circulate and age in blood and are rapidly cleared by the hepatic Ashwell-Morell receptor (AMR). And others have shown, in a series of studies using genetically modified mice or pharmacological inhibitors that platelets undergo apoptosis by triggering the intrinsic mitochondrial apoptotic machinery. Here, we investigate if desialylation and apoptosis are related events. First, using a newly developed state-of-the-art technique called dynamic BH3 profiling (DBP), we investigated the mitochondria readiness to undergo apoptosis on platelets derived from WT and AMR deficient (Asgr2-/-) mice. In our assay, digitonin-permeabilized platelets were exposed to activators signaling peptides (such as Bim, Bid and PUMA), and as cells undergo apoptosis due to peptide treatment, they released Cytochrome C. Our data showed that desialylated platelets derived from Asgr2-/-mice have high background levels of Cytochrome C release when compared to WT platelets in the presence of all activator peptides, indicating that desialylated platelets are highly primed to apoptosis. We also tested the level of dependence on pro-survival protein, by using sensitizer peptides (Bad, Hrk and MS1). We observed that desialylated platelets (Asgr2-/-platelets), and to a certain degree, WT platelets, are extremely sensitive to BCL-xL inhibition, as indicated by the extremely high response to Bad and Hrk peptides even at lower concentrations (0.1 and 1uM). Surprisingly, WT and Asgr2-/-platelets show very little response to the MS1 peptide, indicating that they are not dependent on MCL1 for survival, as otherwise suggested. Flow cytometry analysis revealed desialylated platelets from Asgr2-/-mice have a ~2-fold increase in Phosphatidylserine (PS) surface exposure when compared to WT platelets. In addition, western blot analysis showed increased expression of cleaved caspase 3 in Asgr2-/-platelets, but no changes in Bcl-xL protein expression between WT and Asgr2-/-platelets. Next, WT and Asgr2-/-mice received a single dose of the BH3 mimetic, ABT-737, which binds and inhibits pro-survivor proteins (Bcl-2, Bcl-xL and Bcl-w) inducing apoptosis in vivo. Approximately 2 hours after the injection of ABT-737, we observed a big drop on platelets counts in both WT (~42%) and Asgr2-/-(~59%) mice. Importantly, platelets from Asgr2-/-mouse were cleared more efficiently (~20%) from the circulation when compared to those in WT mice, consistent with the ~20% increment in platelet number observed in this mouse model and supporting the notion that the platelets that circulate longer in the Asgr2-/-mice are more sensitive to apoptotic events. To investigate if apoptosis could be triggering platelet desialylation, WT mice were treated with ABT-737 and 1hour later (time point before platelet count drop), platelets were collected and analyzed by flow cytometry. Interestingly, analysis of galactose exposure by RCA-I lectin showed no differences in desialylation between ABT-737 and PBS control groups. On the other hand, Phosphatidylserine (PS) exposure was significantly elevated on ABT-737 group, indicating that platelets were undergoing apoptosis without changing their sialylated status. To confirm our in vivodata, freshly isolated washed WT platelets were treated with ABT-737 to induce apoptosis or Neuraminidase (NA) to desialylated platelets. NA treatment induced platelet desialylation (increased RCA-I binding) in WT platelets, as expected, and interestingly triggered apoptosis, judge by increased PS exposure in both ABT-737 and NA treated groups. However, ABT-737 treatment wasn't able to induce desialylation as levels of RCA-I binding to platelets was the same when compared to PBS control platelets. Taken together, our data shows that desialylated platelets in circulation are prone to apoptosis. In addition, our findings strongly support the hypothesis that desialylation of platelet surface glycoproteins trigger the intrinsic apoptotic pathway in platelets in vivo. Disclosures No relevant conflicts of interest to declare.

scholarly journals Rare event selection of fetal nucleated erythrocytes in maternal blood by flow cytometry

Cytometry ◽  
1996 ◽  
Vol 23 (3) ◽  
pp. 218-227 ◽  
Author(s):  
Dorothy E. Lewis ◽  
Wendy Schober ◽  
Sarah Murrell ◽  
Dianne Nguyen ◽  
Jeffrey Scott ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Rare Event ◽  
Maternal Blood ◽  
Event Selection ◽  
Selection Of
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