an-optimized-protocol-to-analyze-glycolysis-mitochondrial-respiration

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

National Institutes of Health

The CD200 receptor (CD200R) is present mainly on myeloid cells and gives inhibitory signals when engaged by its ligand CD200. The interaction is currently of therapeutic interest in cancer and inflammation. However functional effects are complicated by the fact that CD200R is itself polymorphic and also a member of a paired receptor family with four closely related gene products in mice called CD200RLa etc. We show that a second allele of CD200R (termed CD200R(2)) that differs in 7 amino acids also binds CD200 but did not react with the widely used CD200R antibody OX110. Biochemical and functional analysis showed that the CD200/CD200R interaction was blocked by the OX131, mAb that recognises both CD200R(1) and CD200R(2), but not by OX110 mAb. Both mAb can give agonistic inhibitory signals but functional analysis shows OX131 mAb also has the potential to block inhibition by preventing the ligand-receptor interaction and hence gives opposing effects. Although OX131 mAb cross-reacts with the activating receptor CD200RLe, it is specific for CD200R in C57BL/6 whilst OX110 mAb cross-reacts on CD200RLc. The results show the importance of the repertoire of paired receptors in strains or individuals and mAb used with implications for paired receptor analysis and therapeutics.

Dissection of agonistic and blocking effects of CD200 receptor antibodies.

Barcoding of biological samples is a commonly used strategy to mark or identify individuals within a complex mixture. However, cell barcoding has not yet found wide use in flow cytometry that would benefit greatly from the ability to analyze pooled experimental samples simultaneously. This is due, in part, to technical and practical limitations of current fluorescent dye-based methods. In this study, we describe a simple, versatile barcoding strategy that relies on combinations of a single Ab conjugated to different fluorochromes and thus in principle can be integrated into any flow cytometry application. To demonstrate the efficacy of the approach, we describe the results of a variety of experiments using live cells as well as fixed and permeabilized cells. The results of these studies show that Ab-based barcoding provides a simple, practical method for identifying cells from individual samples pooled for analysis by flow cytometry that has broad applications in immunological research.

Billur Akkaya

Laboratory of Immunology,
National Institute of Allergy and Infectious Diseases,
Laboratory of Immunology, National Institute of Allergy and Infectious Diseases

National Institutes of Health

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Billur Akkaya

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory of Immunology,National Institute of Allergy and Infectious Diseases,Laboratory of Immunology, National Institute of Allergy and Infectious Diseases

National Institutes of Health

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Laboratory of Immunology,
National Institute of Allergy and Infectious Diseases,
Laboratory of Immunology, National Institute of Allergy and Infectious Diseases