Carl Simard

Urea substitutes toxic formamide as destabilizing agent in nucleic acid hybridizations with RNA probes

Since their introduction some three decades ago, methods for hybridization analysis of nucleic acids immobilized on solid supports have evolved to improve the sensitivity, speed, and convenience of their application. However, in many cases these methods still require the use of solutions containing formamide, a recognized hazardous solvent with potential toxicity. Here, we have compared the efficiency of urea to that of formamide as denaturing agent in nucleic acid hybridization with RNA probes. We show that urea at concentrations of 2–4 molar in solution performs as good as 50% formamide to reduce heterologous background hybridization in Northern blotting experiments realized at 68°C. Presence of urea at higher concentrations resulted in reduced hybridization sensitivity, possibly due to increased viscosity. When tested in Southern blot analysis of genomic DNA, our results revealed that the use of urea in hybridization solution is also suitable to carry out single‐copy gene detection. Together, these findings show that urea can efficiently and safely replace formamide in solutions.

A global look into human T cell subsets before and after cryopreservation using multiparametric flow cytometry and two-dimensional visualization analysis.

The cryopreservation of human lymphocytes is an essential step for the achievement of several cellular therapies. Besides, T cells are considered as promising actors in cancer therapy for their cytotoxic and regulatory properties. Consequently, the development of tools to monitor the impact of freezing and thawing processes on their fine distribution may be an asset to achieve quality control in cellular therapy. In this study, the phenotypes of freshly isolated human mononuclear cells were compared to those observed following one cycle of cryopreservation and rest periods 0h, 1h and 24h after thawing but before staining. T cells were scrutinized for their distribution according to naive, memory effector, regulatory and helper subsets. Flow cytometry analyses were done using eight-color antibody panels as proposed by the Human Immunophenotyping Consortium. Data were further analyzed by using conventional directed gating and clustering software, namely SPADE and viSNE. Overall, SPADE and viSNE tools were very efficient to monitor the outcome of PBMC populations and T cell subsets. T cells were more sensitive to cryopreservation than other cells. Our results indicated that submitting the thawed cells to a 1h rest period improved the detection of some cell markers when compared to fresh samples. In contrast, cells submitted to a 24h rest period, or to none, were less representative of fresh sample distribution. The heterogeneity of PBMC, as well as the effects of freeze-thaw cycle on their distribution, can be easily monitored by using SPADE and viSNE.

Feasibility study: Phosphospecific flow cytometry enabling rapid functional analysis of bone marrow samples from patients with multiple myeloma

Multiple myeloma (MM) is an incurable cancer accounting for about 2% of cancer deaths. Its diagnosis is based on a combination of criteria, which are not always easily measurable. Flow cytometry now allows multiplex analysis of intracellular signaling at the single cell level. We investigated the feasibility of using intracellular protein phosphorylation analysis by flow cytometry on primary plasma cells from bone marrow and its usefulness in MM diagnosis.

Implementing a routine flow cytometry assay for nucleated red blood cell counts in cord blood units

As required by standards organizations, Héma‐Québec Cord Blood Bank performs enumeration of nucleated red blood cells (NRBCs) in cord blood units (CBUs). This study presents the validation and implementation approaches developed to transfer the routine NRBC enumeration from the manual blood film method to a flow cytometric assay.

Rapid determination of il-6 specific activity by flow cytometry

Current methods to measure the specific activity of cytokines are based on the time-consuming determination of the growth curve of a sensitive cell line. Here, we present a faster alternative based on flow cytometry, by determining the dose-response curve of cellular response to a cytokine. By using World Health Organization (WHO) cytokine standards, rapid determination of cytokine specific activity is now possible, as it takes only a few hours to achieve, in comparison to days with the classical method thus allowing laboratories to rapidly and easily assess the potency of their cytokines.

In Vitro Culture of Human Hematopoietic Stem Cells in Serum Free Medium and Their Monitoring by Flow Cytometry.

Hematopoietic stem cells can be isolated from human blood cells trapped in leukoreduction systems. The leukoreduction systems filters or chambers are usually discarded from routine blood or platelet donations in blood banks around the world. These CD34+ cells are a good source of normal stem cells and can be used as models to characterize the blood stem cells before and after culture in vitro. This chapter contains detailed methodologies for the isolation of stem cells from peripheral blood, the culture of these cells in a medium exempt of animal proteins and for the flow cytometry analysis of the resulting cell population for the characterization of their differentiation.