Flow cytometry is a multi-parameter, rapid and efficient method for qualitative analysis and quantitative determination of various fluorescently labeled particles in liquid flow. Flow cytometry has been applied in multiple disciplines such as immunology, virology, molecular biology, cancer biology and infectious disease monitoring. However, the application of flow cytometry in plant research is hampered due to the special composition and structure of plant tissues and cells, such as cell walls and secondary metabolites. In this paper, the development, composition and classification of flow cytometry were introduced. Subsequently, the application, research progress and application limitations of flow cytometry in plant field were discussed. At last, the development trend of flow cytometry in plant research was prospected, which provides new perspectives for broadening the potential application scope of plant flow cytometry.
Flow Cytometry/methods* ; Plants ; Fluorescent Dyes

BACKGROUND: The importance of the determination of cell viability has prompted the development of several assays of viability that utilize the exclusion of certain dyes by viable cell membranes. Recently, flow cytometry has been adapted to estimate cell viability by using fluorescent dye which is excluded by living cells on the basis of altered dead cell properties. OBJECTIVE: We have developed a flow Cytometric method for measuring cell viability after staining with propidium iodide (PI) and have compared it with the classical colorimetric method, MTT assay, which is currently widely used in cytotoxicity assays in the research field. METHODS: We performed flow cytometry and MTT assay for the comparison of the sensitivity of the assessment of cell viability. RESULTS: Decrease of cell viability was measured by flow cytometry with the addition of as little as 0.002% Triton-X 100 in comparison to MTT assay which could only reveal a similar decrease of cell viability with the new method to 0.008% Triton-X 100. CONCLUSION: Our results demonstrate this new method to be more sensitive and simple for the assessment of cell viability.
Cell Membrane ; Cell Survival* ; Coloring Agents ; Flow Cytometry ; Methods* ; Propidium*

OBJECTIVETo prepare stable chicken red blood cells for the calibration of flow cytometry.

METHODSThe traditional isolation method of chicken red blood cells was modified by incorporating gelatin technique, Ca2+-free HBSS treatment and low-speed centrifugation. The effect of fluorescence staining of the cells was improved by the addition of TritonX-100 to enhance the membrane permeability and Rnase enzymes to disintegrate RNA tiles. The modified method was compared with the traditional method for viability of the freshly isolated cells and the DNA content coefficient of variation (CV) of the fixed cells.

RESULTSChicken red blood cells obtained by the modified method showed a significantly higher viability than those obtained by the traditional method [(98.5∓3.5)% vs (93.5∓2.7)%, P<0.05]. After glutaraldehyde fixation, the isolated cells with the modified method were stable during the 90-day preservation with a significantly lower CV than the cells obtained by the traditional method [(6.0∓0.3)% to 6.2∓0.4% vs (8.6∓0.5)% to (13.1∓1.4)%, P<0.01].

CONCLUSIONThe chicken red blood cells isolated using the modified method can be applicable for calibration of flow cytometry.

Animals ; Calibration ; Chickens ; Erythrocytes ; cytology ; Flow Cytometry ; instrumentation ; methods

Bone marrow morphology and karyotyping are effective ways to diagnose myelodysplastic syndromes. However, there are still many patients without these two abnormalities. Recently, there are considerable amount of evidence showing that flow cytometric immunophenotyping is a good approach to detect the abnormal differentiation and maturation of different cell lines and maturation stages of bone marrow cells in MDS patients. The highly reproducible results allow to distinguish MDS from other non-clonal hematocytopenia disorders and have a diagnostic advantage especially in case of low-risk MDS. Meanwhile, it showed a good correlation with the morphology and karyotyping, and with the International Prognostic Scoring System (IPSS) playing a prognostic role. This article reviewed recent advances of research in this field such as the application of FCM in MDS detection, the correlation of FCM parameters with morphologic and cytogenetic diagnosis of MDS as well as the relationship of parameters with typing and prognosis of MDS and so on.
Flow Cytometry ; methods ; Humans ; Immunophenotyping ; Myelodysplastic Syndromes ; diagnosis ; immunology

Antigens, CD34 ; Cell Count ; methods ; Flow Cytometry ; Humans

In vitro compartmentalization (IVC) links genotype and phenotype by compartmentalizing individual genes (including expression system) or cells into a micro-droplet reaction region. Combined with fluorescence-activated cell sorting (FACS), it can detect and separate single droplets in ultra-high throughput. IVC-FACS screening method has been widely used in protein engineering, enzyme directed evolution, etc. However, it is difficult to control the homogeneity of droplet size by mechanical dispersion method in previous studies, which seriously affects the quantitative detection of droplets and reduces the efficiency and accuracy of this screening method. With the rapid development of microfluidic chip manufacturing technology, the microfluidic chip-based methods for droplet generation are becoming more efficient and controllable. In this study, firstly, the water-in-oil (W/O) single-layer droplet generation chip was used to prepare single-layer monodisperse W1/O droplets at a high generation frequency, and then the W1/O droplets were reinjected into water-in-oil-in-water (W/O/W) double-layer droplet generation chip to prepare uniform W1/O/W2 double-layer emulsion droplets. By optimizing the flow rate and ratio of the oil and water phases, a single-layer micro-droplet can be generated with a diameter range from 15.4 to 23.2 μm and remain stable for several days under normal incubation. Then the single-layer droplets were reinjected into the double emulsion generation chip. By adjusting the flow rate of drop phase, oil phase and water phase, the double-layer emulsion droplets with a diameter range from 30 to 100 μm at a rate of 1 000 droplets/s could be obtained. Escherichia coli embedded in the double-layer emulsion droplets could be cultured and induced for protein expression. This study lays a foundation for the establishment of a high-throughput screening method based on the droplet and flow cytometry.
Emulsions ; Flow Cytometry ; High-Throughput Screening Assays ; Microfluidics ; methods

Orientia tsutsugamushi causes scrub typhus, which is endemic in many countries in the Asia-Pacific region including Korea. Recent emergence of doxycycline-resistant strains from Thailand has underlined the importance of the susceptibility tests of O. tsutsugamushi to antibiotics. To improve the flow cytometric technique for the susceptibility test, we applied a monoclonal antibody (MAb) in the quantification of O. tsutsugamushi. With using MAb FS15, we determined the doxycycline susceptibility of two strains, Boryong and AFSC-4 strain which is reported to be doxycycline-sensitive and resistant, respectively. The growth of both strains was inhibited to below 10% of the control in the presence of 0.1 microgram/mL or higher concentrations of doxycycline. We suggest that our approach is more quantitative and reproducible than the conventional microscopic methods.
Orientia tsutsugamushi/*drug effects/growth & development ; Microbial Sensitivity Tests/*methods ; Flow Cytometry/*methods ; Antibodies, Monoclonal/*immunology

OBJECTIVETo establish an effective method for haploid spermatid enrichment by Hoechst 33342 staining and flow sorting.

METHODSMouse testicular monoplast suspension was prepared by two-step enzyme digestion, and the cells were incubated in the medium containing Hoechst 33342 and Verapamil. Haploid spermatids were separated and enriched according to their DNA content by flow sorting. The gene expressions in the spermatids of several histone-modified enzymes, including the histone acetylases (HAT) and histone deacetylases (HDAC), were examined by RT-PCR and compared with that in the HAT-inhibitor curcumin-treated counterparts.

RESULTSWe successfully enriched the haploid spermatids with high purity and further purified the round and elongated spermatids. RT-PCR results indicated the specificity of the expression of the HAT gene in the spermatids, and that it was influenced by curcumin.

CONCLUSIONFlow sorting can efficiently improve the purity of haploid spermatid enrichment, which helps a lot to elucidate the mechanisms of spermiogenesis.

Animals ; Cell Separation ; methods ; Flow Cytometry ; methods ; Haploidy ; Male ; Mice ; Mice, Inbred ICR ; Spermatids ; cytology

In order to explore the factors that affect CD62p expression in platelet during the whole course of cryopreservated platelets preparation, CD62p expression of platelet was evaluated by flow cytometry assay. The whole course of cryopreservated platelets preparation in order included whole blood collection, centrifugation for fresh platelet-rich plasma preparation, addition of dimethyl sulfoxide, and freeze in -80 degrees C refrigerator and thaw in 38 degrees C water bath. Result showed that the CD62p expressed slowly from whole blood collection to addition of dimethyl sulfoxide, but expressed abruptly after freeze and thaw and it occupied 82 per cent of the whole expression. It was concluded that the whole blood collection, centrifugation for fresh platelet enriched plasma preparation and addition of dimethyl sulfoxide were optimized in the whole course, but the damage to platelets in the whole course of cryopreservation could not be avoided. It suggests that improved techniques are needed to reduce the damage to cryopreservated platelets.
Blood Platelets ; metabolism ; Blood Preservation ; methods ; standards ; Cryopreservation ; methods ; standards ; Flow Cytometry ; Humans ; P-Selectin ; biosynthesis

OBJECTIVETo establish a method for the reliable isolation and culture of infantile hemangioma endothelial cells (HemECs) in vitro.

METHODSProliferative hemangioma specimens were digested by collagenase to form a single cell suspension. The HemECs were isolated using anti-CD31 coated dynabeads. The CD31+ cells were grown in fibronectin coated dishes. HemECs were identified by morphological characteristics and immunocytochemistry. The cells were also examined for their ability to intake LDL.

RESULTSThe method enabled the rapid isolation of HemECs that demonstrated typical endothelial cobblestone morphology in culture. The cells were positively stained for CD31, vWF. They also were labeled with DiI-Ac-LDL.

CONCLUSIONSThis technique can effectively isolate endothelial cells from the proliferative hemangiomas. These cells could be further used to research the mechanism of proliferation and degeneration of infantile hemangioma.

Cell Culture Techniques ; methods ; Cell Line, Tumor ; Endothelial Cells ; cytology ; Flow Cytometry ; Hemangioma, Capillary ; Humans