Electrical properties of living cells have been proven to play significant roles in understanding of various biological activities including disease progression both at the cellular and molecular levels. Since two decades ago, many researchers have developed tools to analyze the cell's electrical states especially in single cell analysis (SCA). In depth analysis and more fully described activities of cell differentiation and cancer can only be accomplished with single cell analysis. This growing interest was supported by the emergence of various microfluidic techniques to fulfill high precisions screening, reduced equipment cost and low analysis time for characterization of the single cell's electrical properties, as compared to classical bulky technique. This paper presents a historical review of single cell electrical properties analysis development from classical techniques to recent advances in microfluidic techniques. Technical details of the different microfluidic techniques are highlighted, and the advantages and limitations of various microfluidic devices are discussed.
Constitutive secretion is predominantly measured by collecting the media from cells and performing plate-based assays. This approach is particularly sensitive to changes in cell number, and a significant amount of effort has to be spent to overcome this. We have developed a panel of quantitative flow cytometry-based assays and reporter cell lines that can be used to measure constitutive secretion. These assays are insensitive to changes in cell number making them very robust and well suited to functional genomic and chemical screens. Here, we outline the key steps involved in generating and using these assays for studying constitutive secretion.
Immunoperoxidase staining was compared with flowcytometry for the enumeration of lymphocyte subsets. The percentages obtained for peripheral blood lymphocytes using immunoperoxidase (CD3 = 76 CD4 = 27.9, B = 10.7 CD4/CD8 = 1.8) differed significantly from those obtained by flowcytometry (CD3 = 65.7 CD4 = 39.4, CD8 = 25.6, B = 16.7, HLA DR = 11.9 CD4/CD8 = 1.54) for certain subsets (CD3, CD4, B). There was no significant difference in lymphocyte subsets between children and adults using the same method. These differences are probably due to the different methods used to prepare lymphocytes for analysis. Other factors that should also be considered are the presence of CD4 antigen on monocytes and CD8 on natural killer cells.
Myelodysplastic syndromes (MDS) are a group of haematological malignancies categorized by ineffective hematopoiesis that result in dysplasia. Although morphological diagnosis is a traditional and standard technique that is used for the diagnosis of MDS, the heterogeneous blood and bone marrow characteristics of MDS patients can potentially obscure the right diagnosis. Thus, we have utilized flow cytometric immunophenotyping as a supportive mechanism to obtain a more accurate and faster method for detection of abnormal markers in MDS. Flow cytometry was used for analyzing bone marrow samples from newly diagnosed MDS patients to investigate the abnormal antigen expression patterns in granulocytic, monocytic, erythroid, lymphoid lineages and myeloid precursors. The results were compared with those obtained from cases that had Idiopathic Thrombocytopenic Purpura (ITP) as a control. The most common abnormality found in the granulocytic lineage was the decrease of CD10. Low expressions of CD13 were the most frequent abnormality in the monocytic lineage. The erythroid lineage was found to have low expression of CD235A+/CD71+, reduce of CD71 and decreased CD235a. In conclusion, this method is useful for confirming cases in which it is difficult to make a diagnosis by morphology.
Several fixation and permeabilization techniques that enable the flow cytometric analysis of the cell contents have been introduced in recent years. These methods allow sensitive detection of intracellular antigens that facilitates the diagnosis of certain diseases. We have undertaken in this study to evaluate a simple method of fixation and permeabilization using 2% paraformaldehyde and Tween 20. Intracellular antigens in three different leukaemia cases were analysed. We found that the method was reliable and easy. Intracellular kappa light chains were found in abundance in a case of plasma cell leukaemia. CD3 and CD22 were found in greater amount intracellularly than on the surface in pre-T-ALL and pre-pre B-ALL respectively.
Microglia are resident macrophages of the central nervous system (CNS). Apart from playing vital roles as sentinel cells, they are crucial in physiological processes such as synaptic pruning during brain development. CNS disorders require an understanding of the contribution of each cellular compartment to the pathogenesis. Elucidating the role of microglia in disease development and progression in the intricate CNS environment is technically challenging and requires the establishment of reliable, reproducible techniques to isolate and culture microglia. A number of different protocols have been developed for isolation of neonatal microglia and here we compare two widely used methods, namely, mild trypsinization and EasySep® magnetic separation. EasySep® magnetic separation provided higher microglia yield, and flow cytometric evaluation of CD11b and F4/80 markers revealed that EasySep® separation method also produced significantly higher purity compared to mild trypsinization. Microglia isolated using EasySep® separation method were functional, as demonstrated by the generation of nitric oxide, IL-6, TNF-α, and MCP-1 in response to lipopolysaccharide stimulation. In summary, this study has revealed that magnetic separation is superior to mild trypsinization in terms of yield and purity of microglia.
Studies show that the pH of the malaria parasite's digestive vacuole (DV) plays a key role in the physiological functions of this organelle and antimalarial drug accumulation, and yet is technically difficult to measure. In this study, a flow cytometry-based technique was developed to measure the DV pH using a ratiometric pH indicator, FITC-dextran loaded into the DV of saponin-permeabilized parasites. To calculate the DV pH, a standard pH calibration curve was generated by incubating the saponin-permeabilized cells in buffers with different pH in the presence of an ionophore, CCCP. The measured average pH of the DV was 5.27 ± 0.03 that is approximately the same in the parasites observed microscopically by Hayward et al. (2006) (5.50 ± 0.14) using the same probe. The removal of glucose from the medium, causing a rapid depletion of parasite ATP, resulted in an alkalization of the DV. The DV was reacidified upon restoration of glucose to the medium. This technique provides a rapid, simple and quantitative measurement of the DV pH on a large number of cells. It will also be useful in future attempts to evaluate the effect of antimalarial drugs (i.e. chloroquine and artemisinin-based drugs) in pH changes of the DV.
Myeloperoxidase (MPO) is present in azurophilic granules which appear in the promyelocyte stage of differentiation and is expressed in granulomonocytic cells. MPO is usually detected by cytochemistry. The demonstration of peroxidase in at least 3% of bone marrow blasts defines an acute leukaemia as acute myeloblastic leukaemia (AML). MPO is important in distinguishing acute myeloblastic leukaemia (AML) from acute lymphoblastic leukaemia (ALL). It is difficult to diagnose AML with minimal evidence of myeloid differentiation (AML- M0) by conventional light microscopy. However, these AML-M0 blasts can be detected by monoclonal antibodies. Anti-MPO recognizes the enzymatically inactive precursor forms of MPO. There are a few commercially available monoclonal antibodies against MPO. In this study, we evaluated two monoclonal antibodies against MPO from different commercial sources.
The aim of this project was to compare dual and tri-colour reagents for lymphocyte immunophenotyping. A total of 37 patient and normal specimens were immunophenotyped concurrently with the following mean values (% dual vs tri-colour): CD3 (69.4 vs 68.3) CD4 (24.0 vs 24.2) and CD19 (13.9 vs 12.6). A comparison of the results obtained using the paired t test showed that there were no significant differences for cells expressing CD3, CD4 and CD19. However, there was a significant difference in the NK (18.3 vs 16.3) cell component. A major advantage in using 3 colour immunophenotyping is the ability to analyse specimens that cannot be analysed using dual colour reagents due to debris or contamination of the gate with non-lymphocytic cells.
Analysis of cancer-derived extracellular vesicles (EVs) in biofluids potentially provides a source of disease biomarkers. At present there is no procedure to systematically identify which antigens should be targeted to differentiate cancer-derived from normal host cell-derived EVs. Here, we propose a computational framework that integrates information about membrane proteins in tumors and normal tissues from databases: UniProt, The Cancer Genome Atlas, the Genotype-Tissue Expression Project, and the Human Protein Atlas. We developed two methods to assess capture of EVs from specific cell types. (1) We used palmitoylated fluorescent protein (palmtdTomato) to label tumor-derived EVs. Beads displaying antibodies of interest were incubated with conditioned medium from palmtdTomato-expressing cells. Bound EVs were quantified using flow cytometry. (2) We also showed that membrane-bound Gaussia luciferase allows the detection of cancer-derived EVs in blood of tumor-bearing animals. Our analytical and validation platform should be applicable to identify antigens on EVs from any tumor type.
Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haemopoietic stem cell disorder arising from somatic mutation of the X-linked PIG-A gene which leads to deficiency of the glycosylphosphatidylinositol (GP1) membrane anchor proteins such as CD 59 (MIRL: membrane inhibitor of reactive lysis) and CD 55 (DAF: decay accelerating factor). Allogeneic peripheral blood stem cell transplant (PBSCT) is a curative mode of treatment in symptomatic PNH patients. Assessment of donor chimerism for PBSCT can be performed by various methods including short tandem repeat loci (STR) and variable number of tandem repeats (VNTR). Flow cytometry, which is much cheaper and faster, also can be used to assess engraftment in patients with PNH. Engrafted patients will show the presence of CD 55 and CD 59 on their red cells and white cells. We describe here the usefulness of flow cytometry in the assessment of donor chimerism following allogeneic PBSCT, in a case of PNH.
The main aim of the present study was to evaluate the capacity of stem cells from human exfoliated deciduous teeth (SHED) to enhance mandibular distraction osteogenesis (DO) in rabbits.
Several techniques have been devised for the dissociation of tissues for primary culture. These techniques can affect the quantity and quality of the isolated cells. The aim of our study was to develop the most appropriate method for the isolation of human umbilical cord-derived mesenchymal (hUCM) cells. In the present study, we compared four methods for the isolation of hUCM cells: three enzymatic methods; collagenase/hyaluronidase/trypsin (CHT), collagenase/trypsin (CT) and trypsin (Trp), and an explant culture (Exp) method. The trypan blue dye exclusion test, the water-soluble tetrazolium salt-1 (WST-1) assay, flow cytometry, alkaline phosphatase activity and histochemical staining were used to evaluate the results of the different methods. The hUCM cells were successfully isolated by all methods but the isolation method used profoundly altered the cell number and proliferation capacity of the isolated cells. The cells were successfully differentiated into adipogenic and osteogenic lineages and alkaline phosphatase activity was detected in the hUCM cell colonies of all groups. Flow cytometry analysis revealed that CD44, CD73, CD90 and CD105 were expressed in all groups, while CD34 and CD45 were not expressed. The expression of C-kit in the enzymatic groups was higher than in the explant group, while the expression of Oct-4 was higher in the CT group compared to the other groups. We concluded that the collagenase/trypsin method of cell isolation yields a higher cell density than the others. These cells expressed a higher rate of pluripotent cell markers such as C-kit and Oct-4, while the explant method of cell isolation resulted in a higher cell proliferation rate and activity compared to the other methods.