Demands and technical developments of clinical flow cytometry with emphasis in quantitative,spectral,and imaging capabilities

As the gold-standard method for single-cell analysis,flow cytometry enables high-throughput and multiple-parameter characterization of individual biological cells.This review highlights the demands for clinical flow cytometry in laboratory hematology(e.g.,diagnoses of minimal residual disease and various types of leukemia),summarizes state-of-the-art clinical flow cytometers(e.g.,FACSLyricTMby Becton Dickinson,DxFLEX by Beckman Coulter),then considers innovative technical improvements in flow cytometry(including quantitative,spectral,and imaging approaches)to address the limitations of clinical flow cytometry in hematology diagnosis.Finally,driven by these clinical demands,future developments in clinical flow cytometry are suggested.

Morphological Effects of Natural Products on Schizosaccharomyces pombe Measured by Imaging Flow Cytometry

Gaining a full understanding of the mechanisms of action of natural products as therapeutic agents includes observing the effects of natural products on cellular morphology,because abnormal cellular morphology is an important aspect of cellular transformations that occur as part of disease states.In this study a set of natural products was examined in search of small molecules that influence the cylindrical morphology of fission yeast Schizosaccharomyces pombe.Imaging flow cytometry of large populations of S.pombe exposed to natural products captured cell images and revealed changes in mean length and aspect ratio of cells.Several natural products were found to alter S.pombe’s morphology relative to control,in terms of elongating cells,shrinking them,or making them more round.These results may facilitate future investigations into methods by which cells establish and maintain specific shapes.

The feasibility of antitumor drugs chemosensitivity testing by flow cytometry

Objective: To investigate the feasibility of chemosensitivity testing of antitumor drugs by flow cytometry in clinical applications so as to provide experimental and theoretical basis for the establishment of a novel antitumor drugs sensitivity testing and the screening of particular antitumor drugs. Methods: Detect the apoptosis rate of 12 cases of Molt-4 cell line, 57 cases of fresh clinical gastrointestinal tumor cells by Sub-G1 and Annexin V assay of flow cytometry under the effects of antitumor drugs at different times and the outcomes were compared with the ones of the MTT (3-(4,5-dimethylthiazolyl-2) -2,5-diphenyltetrazolium bromide) assay. Results: The lethality of drugs on Molt-4 cell, clinical gastrointestinal tumor cells had a positive correlation with the acting time of antidrugs by employing Annexin V, Sub-G1 and MTT assay. Drug-incurring maximum lethality of Annexin V assay was higher than MTT colorimetric assay, that of Sub-G1 was lower than MTT assay, the virtual times of Annexin V and Sub-G1 assay were obviously earlier than that of MTT colorimetric assay. Conclusion: Annexin V and Sub-G1 assay of flow cytometry can be taken as potent protocols testing anti-tumor drug chemosensitivity. Annexin V assay is featured by more sensitive, concise, reliable compared with the classical chemosensitivity testing assay of MTT colorimetric assay and it possesses clinical applied value.

Flow Cytometric Evidence for Hydroxyl Radical-induced Apoptosis in Tobacco Protoplasts

Protoplasts prepared from tobacco (Nicotiana tabacum L., cultivar BY-2) suspension cells have similar morphological characteristics to those in animal cells. The hallmarks of apoptosis such as condensation and peripheral distribution of nuclei, TUNEL positive reaction, and DNA ladders were observed when tobacco protoplasts were treated with the hydroxyl radical generating system (1.0 mmol/L FeSO4/0.5 mmol/L H2O2). In animals, the loss of transmembrane potential (DeltaPsi(m)) and the exposure of phospholipid phosphatidylserine (PS) are believed to be the main apoptosis events. To test whether these significant processes take place in plants, flow cytometry was used to detect annexin V binding and changes in DeltaPsi(m). Results showed that the PS turned out from inner membrane and DeltaPsi(m) gradually decreased during the apoptosis. All these apoptotic characteristics proved that hydroxyl radicals can cause typical programmed cell death (PCD) in tobacco protoplasts and this design can be served as an effective experiment system to explore the mechanism of plant apoptosis.

THE EFFECTS OF LOW CONCENTRATIONS OF ZINC ON ETOPOSIDE INDUCED HL-60 CELLS APOPTOSIS

Objective Observation of the effects of lower concentrations of zinc (20 ～ 400 μmol /L), which is nearby the physiological concentration, on etoposide induced HL-60 cells apoptosis. Methods Us-ing the flow cytometry, DNA extraction, electrophoresis, and fluoresence microscopic observation. Results We demonstrated that The low concentrations of zinc also affect cells apoptosis. If zinc was added at early time, even 200μmol/L could inhibit V16 induced HL-60 cell apoptosis completely in 4h. But at this concentration, zinc also seems to have cell toxicity, not prolong the time of protection effect. Conclusion Zinc plays mul-tiple roles in cellular functions and in protecting cells from exogenous deleterious factors. Zinc plays a complex, dose- and time-dependent role in apoptosis.

Gender difference in the neuroprotective effect of rat bone marrow mesenchymal cells against hypoxiainduced apoptosis of retinal ganglion cells

Bone marrow mesenchymal stem cells can reduce retinal ganglion cell death and effectively prevent vision loss. Previously, we found that during differentiation, female rhesus monkey bone marrow mesenchymal stem cells acquire a higher neurogenic potential compared with male rhesus monkey bone marrow mesenchymal stem cells. This suggests that female bone marrow mesenchymal stem cells have a stronger neuroprotective effect than male bone marrow mesenchymal stem cells. Here, we first isolated and cultured bone marrow mesenchymal stem cells from female and male rats by density gradient centrifugation. Retinal tissue from newborn rats was prepared by enzymatic digestion to obtain primary retinal ganglion cells. Using the transwell system, retinal ganglion cells were co-cultured with bone marrow mesenchymal stem cells under hypoxia. Cell apoptosis was detected by flow cytometry and caspase-3 activity assay. We found a marked increase in apoptotic rate and caspase-3 activity of retinal ganglion cells after 24 hours of hypoxia compared with normoxia. Moreover, apoptotic rate and caspase-3 activity of retinal ganglion cells significantly decreased with both female and male bone marrow mesenchymal stem cell co-culture under hypoxia compared with culture alone, with more significant effects from female bone marrow mesenchymal stem cells. Our results indicate that bone marrow mesenchymal stem cells exert a neuroprotective effect against hypoxia-induced apoptosis of retinal ganglion cells, and also that female cells have greater neuroprotective ability compared with male cells.

The approaches in detecting cell cycle specificity of Fas-mediated apoptosis in leukemia cell lines and activated PBLs in vitro

Objective： To establish a system in detecting the cell cycle specificity induced by recombinant human Fas ligand in vitro, so as to provide a reliable platform for further exploring the mechanism of cell cycle control and regulation in Fas-mediated apoptosis. Methods： The target cells-leukaemia cell lines and activated peripheral blood lymphocytes stimulated by phytohemagglutinin were incubated with recombinant human Fas ligand for 6 to 36 h, apoptosis was detected by sub-G1, common annexin-Ⅴ/PI and modified annexin Ⅴ and propidium iodide （API） methods and analysed by flow cytometry. Results： The modified API method demonstrated that Fas-mediated apoptosis was cell cycle specific and initiated at G1 phase. The common annexinⅤ/PI method showed the most appropriate condition for the detection of typical cell cycle-specific apoptosis. The sub-G1 method could only illuminate late apoptosis and DNA histogram. Conclusion： Fas-mediated apoptosis was cell cycle-specific and initiated at G 1 phase. Based on the modified API and common AnnexinⅤ/PI methods, the establishment of stable and typical cell cycle-specific model in Fas-mediated apoptosis in vitro was feasible.

The Mechanism of Cisplatin-induced Apoptosis in HeLa Cells

OBJECTIVE To study the mechanism of apoptosis induced by cisplatin in vitro in HeLa cells cervical cancer cell line. METHODS The inhibitory effect of cisplatin on HeLa cell growth was analyzed by the MTT assay. Cell apoptosis was measured with flow cytometry and Hoechst 33258 staining following cisplatin treatment. The effect of cisplatin on transcription of HPV E6 was analyzed by RT-PCR and protein expression of E6, P53, p21, Bax and Bcl-2 was studied by Western blots. RESULTS Cisplatin inhibited cellular proliferation in a time and dose- dependant manner. The sub-G1 peak by flow cytometry showed a higher apoptotic rate in the experimental group compared to the controls and Hoechst 33258 staining indicated that apoptosis was induced by cisplatin. Results of RT-PCR demonstrated that cisplatin decreased tran- scription of E6. Western Blots showed that cisplatin decreased protein expression of E6 and increased protein expression of P53, p21 and Bax but had no effect on protein expression of Bcl-2. CONCLUSION Cisplatin induces apoptosis and death of HeLa cells through the suppression of HPV E6 and restoration of p53 function.

Effects of 60 Hz electromagnetic field exposure on testiculargerm cell apoptosis in mice

Aim: To evaluate the effects of 60 Hz extremely low frequency (ELF) elelctromagnetic field (EMF) exposure on germ cell apoptosis in the testis of mice. Methods: Adult male BALB/c mice (7 weeks of age) were exposed to a 60 Hz EMF of 0.1 mT or 0.5 mT for 24 h/day. A sham-exposed group served as the control. After 8 weeks of exposure, the mice were sacrificed. Germ cell apoptosis in the testis was assessed by histopathological examination, the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) and flow cytometric examination of isolated spermatogenic cells stained with 7 aminoactinomycin D (7-AAD). Results: EMF exposure did not significantly affect the body and testis weights, but significantly increased the incidence of germ cell death. The distinguishing morphological feature of EMF exposure was a decrement in the number of well organized seminiferous tubules. Quantitative analysis of TUNEL-positive germ cells showed a significantly higher apoptotic rate in the 0.5 mT exposed mice than that in the sham controls (P<0.05), while the difference between the two exposed groups was insignificant. The TUNEL-positive cells were mainly spermatogonia. In flow cytometry analysis, the percentage of live cells [forward scatter count (FSC)high7-AAD-] was lower in the exposed groups than that in the controls (Figure 5A), but the decrease in viability was not statistically significant. Conclusion: Continuous exposure to ELF EMF may induce testicular germ cell apoptosis in mice.

Early apoptosis in intestinal and diffuse gastric carcinomas

INTRODUCTIONApoptosis,described by Kerr in 1972,plays a keyrole in all types of regulated cellular processes inmulticellular,organisms.It is defined as amorphologic change,including fragmentation of theDNA,cell shrinkage,dilation of the endoplasmaticreticulum,cell fragmentation and formation

Relationship between Fas/ FasL expression and apoptosis of colon adenocarcinoma cell lines

INTRODUCTIONFas/ FasL system has been identified as a keymediator of apoptosis in tumor cells[1-4]. Theoccurrence and development of neoplasm are closelyrelated to apoptosis[5-7] Most chemotherapeuticdrugs kill cancer cells mainly by inducingapoptosis[8-14].'

Aquaporin-1 down regulation associated with inhibiting cell viability and inducing apoptosis of human lens epithelial cells

AIM：To investigate the role of Aquaporin-1（AQP-1）in lens epithelial cells（LECs）and its potential target genes.AQP-1 is specifically expressed in LECs of eyes and is significant for lens homeostasis and transparency maintenance.Herein,AQP-1 expression in LECs was investigated to evaluate its influence on cell survival in association with its potential role in cataract formation.·M ETHODS：LECs were transfected with lentivirus carrying AQP-1 small interfering RNA（si RNA）.Real-time polymerase chain reaction（PCR）and Western blotting were conducted to detect AQP-1 expression in LECs from different groups.Meanwhile,cell counting kit-8（CCK-8）assay and flow cytometry were performed to measure LEC proliferation and apoptosis,respectively.·RESULTS：AQP-1 expression was significantly reduced in LECs,both at m RNA and protein levels（〈0.05）,after si RNA treatment.Decreased cell viability was detected by CCK-8 assay in LECs with si RNA interference,compared to control cells（〈0.05）.The apoptosis rate significantly increased in cells after si RNA interference（〈0.05）.·CONCLUSION：The decreased cell viability following AQP-1 down regulation is largely due to its induction of apoptosis of LECs.AQP-1 reduction might lead to changes of physiological functions in LECs,which might be associated with the occurrence and development of cataracts.

DETECTION OF B LYMPHOMA CELLS UNDERGOING APOPTOSIS BY ANNEXIN-V ASSAY

To quantitatively analyze apoptotic and secondary necrotic cells unde r apoptosis conditions. Methods. The cells of Burkitt lymphoma (BL) cell line Raji were incubated with 1 .0 μmol/L dexamethasone (DEX) for 2, 4 and 8 h respectively, then stained with Annexin V FITC (fluorescein isothiocyanate conjugated) which was used to detec t the exposed phosphatidylserine (PS) on the epimembrane resulting from a loss o f phospholipid asymmetry in the early stage of apoptosis, and also stained with propidium iodide (PI) which allows analysis of secondary necrotic cells related with cell membrane and DNA damage that probably represent late stage of apoptosi s, then apoptotic cells were quantified by flow cytometry (FCM). Furthermore, An nexin+/PI and Annexin+/PI+ cells were sorted by fluoresence activated cell sorter (FACS), and identified by electron microscopy (EM) and DNA gel electroph oresis. Results. The percentage of apoptotic cells was found to increase with the incuba tion time (r=0.97). This method was sensitive with low detection limit (0.02%), and was reproducible with low coefficient variance (CV)(4.2%). Meanwhile, the Annexin+/PI and Annexin+/PI+ cells were identified as apoptotic and necroti c cells under EM, and DNA extracted from the Annexin+/PI cells was characteri zed by “ladder pattern”. Conclusions. Annexin V assay is a specific, sensitive, accurate, reproductive and quantitative method for analyzing apoptotic cells.

Autophagy occurs within an hour of adenosine triphosphate treatment after nerve cell damage:the neuroprotective effects of adenosine triphosphate against apoptosis

After hypoxia, ischemia, or inflammatory injuries to the central nervous system, the damaged cells release a large amount of adenosine triphosphate, which may cause secondary neuronal death. Autophagy is a form of cell death that also has neuroprotective effects. Cell Counting Kit assay, monodansylcadaverine staining, flow cytometry, western blotting, and real-time PCR were used to determine the effects of exogenous adenosine triphosphate treatment at different concentrations （2, 4, 6, 8, 10 mmol/L） over time （1, 2, 3, and 6 hours） on the apoptosis and autophagy of SH-SY5Y cells. High concentrations of extracellular adenosine triphosphate induced autophagy and apoptosis of SH-SYSY cells. The enhanced autophagy first appeared, and peaked at 1 hour after treatment with adenosine triphosphate. Cell apoptosis peaked at 3 hours, and persisted through 6 hours. With prolonged exposure to the adenosine triphosphate treatment, the fraction of apoptotic cells increased. These data suggest that the SH-SY5Y neural cells initiated autophagy against apoptosis within an hour of adenosine triphosphate treatment to protect themselves against injury.

EFFECTS OF CURCUMIN ON PROLIFERATION AND APOPTOSIS OF HUMAN CERVICAL CARCINOMA HeLa CELLS IN VITRO

Objective: To investigate the regulatory effect of curcumin on proliferation and apoptosis in human cervical carcinoma cell line HeLa in vitro. Methods: Human cervical carcinoma cell line Hela was cultured in vitro. HeLa cells were treated with 10~50 mmol/L curcumin for 24~72 h and the growth inhibition rates of HeLa cells were measured by MTT method. Cell apoptosis was inspected by electron microscopy. In addition, the expression of bcl-2, bcl-xl and caspase-3 protein in HeLa cell were observed by SP immunohistochemistry. Results: Curcumin inhibited the proliferation of HeLa cells on a dose-depending manner. Peak of subG1 appeared on DNA histogram in FCM. A portion of the cells presented the characteristic morphological changes of apoptosis under the electron microscope. The bcl-2, bcl-xl expression was decreased while Caspase-3 expression was increased. Conclusion: Curcumin could significantly inhibit the growth of HeLa cells; inducing apoptosis through up-regulating Caspase-3 and down-regulating expression of bcl-2 and bcl-xl was probably one of its molecular mechanisms.

Relationship between apoptosis and invasive and metastatic potential of hepatocellular carcinoma

Objective: To study relationship between apoptosis and invasive and metastatic potential of hepatocellu- lar carcinoma (HCC). Methods: Apoptotic rate (AR), proliferative index (PI) and S-phase fraction (SPF) were measured by flow cytometry, and p170, p21 and nucleoside diphosphate kinase (ndpk) by strept avidin-biotin complex immunohistochemical technique in 57 pa- tients with HCC. Results: In this group, AR was 1.77% ±0.19%, SPF 12.55% ±0.68%, and PI 20.91% ±1.12% (r =-0.173). p170, p21 and ndpk positive rates were 61.36%, 68, 18%, 52.27% respectively in patients with a mean AR of ≤1.77%, and 23.08%, 38.46%, 84.62% respectively in patients with a mean AR of >1.77% (all P<0.05). In patients with positive tumor invasiveness and metastasis, nd- pk (+) was 43.75%, p21 (+) 75.00%, p170 (+) 65.63%, AR 1.12% ±0. 16%, PI 23.78% ±1.48%, and SPF 13.90 % ±0.99 %. In patients with negative invasiveness and metastasis, however, ndpk (+) was 80.00%, p21 (+) 44.00%, p170 (+) 36.00%, AR 2,32%±0.52%, PI 18.53% ±0.82% and SPF 11.43% ±0.70%. Conclusion: Apoptosis of HCC is negatively correla- ted with its invasive and metastatic potential or other factors as proliferative activity, p21, p170 and ndpk.

Growth Inhibition and Apoptosis Induction in Human Hepatoma Cells by Tanshinone Ⅱ_A

In order to .study the effect of tanshinone ⅡA on growth and apoptosis in human hepatoma cell line BEL-7402 in vitro, the human hepatoma cell line BEL-7402 was treated with tanshinone ⅡA at various concentrations for 72 h. Growth suppression was evaluated by MTT assay; apoptosis-relat-ed alterations in morphology and biochemistry were ascertained under cytochemical staining (Hoechst 33258), transmission electron microscopy (TEM), and DNA agarose gel electrophoresis. Apoptotic rate was quantified by flow cytometry (FCM). The results showed that Tanshinone ⅡA could inhibit the growth of hepatoma cells in a dose-dependent manner, with IC50 value being 6. 28μg/ml. After treatment with 1-10μg/ml tanshinone ⅡA for 72 h, BEL-7402 cells apoptosis with nuclear chro-matin condensation and fragmentation as well as cell shrinkage and the formation of apoptotic bodies were observed. DNA ladder could be demonstrated on DNA electrophoresis. FCM analysis showed hypodiploid peaks on histogram, and the apoptotic rates at μg/ml concentration for 12 h> 24 h, 36 h, 48 h and 72 h were (2. 32±0. 16)%, (3. 01±0. 35) %, (3. 87±0. 43)%, (6. 73±0. 58)% and (20. 85 ± 1. 74) % respectively, which were all significantly higher than those in the control group (1. 07±0. 13) %. It is concluded that Tanshinone ⅡA could induce human hepatoma cell line BEL-7402 apoptosis, which may be related to the mechanism of growth inhibition.

Suppression of renal cell proliferation, induction of apoptosis and cell cycle arrest: Cytotoxicity of vanadium in broilers

The aims of this study were to clarify the effects of high vanadium on the renal cell cycle and apoptosis in broilers. 420 one-day-old avian broilers were divided into six groups and fed on a control diet (vanadium 0.073 mg/kg), and five high vanadium diets (vanadium 5 mg/kg, high vanadium group I;15 mg/kg, high vanadium group II;30 mg/kg, high vanadium group III;45 mg/kg, high vanadium group IV;60 mg/kg, high vanadium group V) throughout the experimental period of 42 days. As tested by flow cytometry, the percentage of apoptotic renal cells was increased in high vanadium group II, III, IV and V when compared with that of control group. The Proliferating index (PI) of renal cell and the ratio of S, G2 + M phase cells were markedly decreased and population of G0/G1 cells was increased in high vanadium group II, III, IV and V. The results showed that dietary vanadium in excess of 15 mg/kg was toxic to kidney by the renal cells cycle arrest and increased apoptosis, which caused the growth depression of the kidney in broilers.

Oxaliplatin and teniposide can inhibit the proliferation and induce the apoptosis of gastric cancer cell line BGC-823 synergistically

Objective:The aim of this study was to investigate the synergistic effects of oxaliplatin and teniposide on proliferation and apoptosis of gastric cancer cell line BGC-823.Methods:MTT assay was carried to examine the inhibition rate of oxaliplatin and teniposide on gastric cancer cell line BGC-823 with various concentrations separately and associatively.The apoptosis rate of BGC-823 cells under the treatment of oxaliplatin or/and teniposide was examined by flow cytometry.The expression level of livin, an apoptosis-associated protein, was explored by western blot.Results:Oxaliplatin or teniposide could remarkably inhibit the BGC-823 gastric cancer cell growth with a dose-response manner, separately and associatively.The inhibition rate of oxaliplatin combined with teniposide on BGC-823 cells was higher than that of single oxaliplatin or single teniposide(P < 0.05), with 0.46 as combination index(CI) value.The apoptosis rates of cells treated by oxaliplatin for 12 h, 24 h and 48 h were 6.13%, 13.86% and 21.48%, respectively, while which of teniposide were 4.60%, 10.72%, 17.07%.But when the two medicines were carried associatively, the apoptosis rates for 12 h, 24 h and 48 h were 11.73%, 24.14% and 44.75%, respectively.Western blot showed that the expression level of livin was more down-regulated when cells were treated by oxaliplatin + teniposide than by oxaliplatin singly.Conclusion:The combination of oxaliplatin and teniposide can exert a synergistic effect on gastric cancer cell BGC-823.

Gambogic acid induces apoptosis and inhibits colorectal tumor growth via mitochondrial pathways

AIM: To investigate the effect of gambogic acid(GA) on apoptosis in the HT-29 human colon cancer cell line. METHODS: H-29 cells were used for in vitro experiments in this study. Relative cell viability was assessed using MTT assays. Cell apoptosis was detected by terminal deoxynucleotidyl transferase d UTP nick end labeling and Hoechst 33342 staining, and quantified by flow cytometry. Cellular ultrastructure was observed by transmission electron microscopy. Real-time PCR and Western blot analyses were used to evaluate gene and protein expression levels. For in vivo experiments, BALB/c nude mice received subcutaneous injections of HT-29 cells in the right armpit. When well-established xenografts were palpable with a tumor size of 75 mm3, mice were randomly assigned to a vehicle(negative) control, positive control or GA treatment group(n = 6 each). The animals in the treatment group received one of three dosages of GA(in saline; 5, 10 or 20 mg/kg) via the caudal vein twice weekly, whereas animals in the negative and positive control groups were given equal volumes of 0.9% saline or 10 mg/kg docetaxel, respectively, via the caudal vein once weekly. RESULTS: The cell viability assay showed that GA inhibited proliferation of HT-29 cells in a dose- and time-dependent manner after treatment with GA(0.00, 0.31, 0.62, 1.25, 2.50, 5.00 or 10.00 μmol/L) for 24, 48 or 72 h. After 48 h, the percentage of apoptotic cells in cells treated with 0.00, 1.25, 2.50 and 5.00 μmol/L GA was 1.4% ± 0.3%, 9.8% ± 1.2%, 25.7% ± 3.3% and 49.3% ± 5.8%, respectively. Ultrastructural analysis of HT-29 cells treated for 48 h with 2.5μmol/L GA revealed apoptotic bodies and condensed and fragmented nuclei. Levels of caspase-8,-9 and-3 m RNAs were significantly increased after treatment with GA(1.25, 2.50 or 5.00 μmol/L) for 48 h(P < 0.05 for all). Protein levels of apoptosis-related factors Fas, Fas L, FADD, cytochrome c, and Apaf-1 were increased in GA-treated cells, whereas levels of pro-caspase-8,-9 and-3 were significantly decreased(P < 0.05 for all). Furthermore, GA significantly and dose-dependently inhibited the growth of HT-29 tumors in a mouse xenograft model(P < 0.05).CONCLUSION: GA inhibits HT-29 proliferation via induction of apoptosis. The anti-cancer effects are likely mediated by death receptor(extrinsic) and mitochondrial(intrinsic) pathways.