Cytogenetic abnormalities are important diagnostic and prognostic criteria for hematologic malignancies. Karyotyping and fluorescence in situ hybridization (FISH) are the conventional methods by which these abnormalities are detected. The sensitivity of these microscopy‐based methods is limited by the abundance of the abnormal cells in the samples and therefore these analyses are commonly not applicable to minimal residual disease (MRD) stages. A flow cytometry‐based imaging approach was developed to detect chromosomal abnormalities following FISH in suspension (FISH‐IS), which enables the automated analysis of several log‐magnitude higher number of cells compared with the microscopy‐based approaches. This study demonstrates the applicability of FISH‐IS for detecting numerical chromosome aberrations, establishes accuracy, and sensitivity of detection compared with conventional FISH, and feasibility to study procured clinical samples of acute myeloid leukemia (AML). Male and female healthy donor peripheral blood mononuclear cells hybridized with combinations of chromosome enumeration probes (CEP) 8, X, and Y served as models for disomy, monosomy, and trisomy. The sensitivity of detection of monosomies and trisomies amongst 20,000 analyzed cells was determined to be 1% with a high level of precision. A high correlation (R2 = 0.99) with conventional FISH analysis was found based on the parallel analysis of diagnostic samples procured from 10 AML patients with trisomy 8 (+8). Additionally, FISH‐IS analysis of samples procured at the time of clinical remission demonstrated the presence of residual +8 cells indicating that this approach may be used to detect MRD and associated chromosomal defects. © 2012 International Society for Advancement of Cytometry

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo. [1][2][3][4][5] Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:1. stem and progenitor cell quiescence, proliferation and/or differentiation [6][7][8] 2. antigen-driven membrane transfer 9 and/or precursor cell proliferation 3,4,10-18 and 3. immune regulatory and effector cell function 1,[18][19][20][21] .Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes 1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins 4,16,18 . Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class [2][3][4]16,18,24 .The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:1. Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies. 2. Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 10 2 -10 3 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used. 3. Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/ dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Parkinson’s disease is a neurodegenerative disorder involving the progressive loss of dopaminergic neurons (DNs), with currently available therapeutics, such as L-Dopa, only able to relieve some symptoms. Stem cell replacement is an attractive therapeutic option for PD patients, and DNs derived by differentiating patient specific stem cells under defined in-vitro conditions may present a viable opportunity to replace dying neurons. We adopted a previously published approach to differentiate Mesenchymal Stem Cells (MSCs) into DN using a 12-day protocol involving FGF-2, bFGF, SHH ligand and BDNF. While MSC-derived DNs have been characterized for neuronal markers and electrophysiological properties, we investigated store-operated calcium entry (SOCE) mechanisms of these DNs under normal conditions, and upon exposure to environmental neurotoxin, 1-methyl, 4-phenyl pyridinium ion (MPP+). Overall, we show that MSC-derived DNs are functional with regard to SOCE mechanisms, and MPP+ exposure dysregulates calcium signaling, making them vulnerable to neurodegeneration. Since in-vitro differentiation of MSCs into DNs is an important vehicle for PD disease modeling and regenerative medicine, the results of this study may help with understanding of the pathological mechanisms underlying PD.

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