Use of both CD63 up regulation and IgE down regulation for the flow cytometric analysis of allergen induced basophil activation. Definition of an activation index

2007 ◽  
Vol 56 (7) ◽  
pp. 291-296 ◽  
Author(s):  
J. Sainte-Laudy ◽  
A. Boumediene ◽  
F. Touraine ◽  
I. Orsel ◽  
C. Brianchon ◽  
...  
Keyword(s):  
Flow Cytometric Analysis ◽  
Basophil Activation ◽  
Down Regulation ◽  
Flow Cytometric ◽  
Definition Of

Biological Characteristics of ZUC3 Antigen Expressed on Rat Bone Marrow Mesenchymal Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4254-4254
Author(s):  
He Huang ◽  
Jing Zheng ◽  
Xiaoyu Lai ◽  
Junli Cao ◽  
Jianling Fan
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Flow Cytometric Analysis ◽  
Osteoblastic Differentiation ◽  
Significant Decline ◽  
Human Mscs ◽  
Down Regulation ◽  
Flow Cytometric ◽  
Marrow Mesenchymal Stem Cells

Abstract Objective: Bone marrow mesenchymal stem cells (MSCs) are widely studied in recent years. As an important part of cell identification, specific surface markers of MSCs have been paid a lot of attention to for long, but no breakthrough as yet. Monoclonal antibodies (McAb) against surface of certain cells have been used to characterize cell lineages. ZUC3, a novel murine McAb was produced by hybridoma technology previously, which was specifically reactive with both human MSCs and rat MSCs. Studying the expression of ZUC3 antigen on rat MSCs after passage and differentiation, it was to define whether ZUC3 antigen would be available for the identification of rat MSCs or their differentiation lineages. Methods: Rat MSCs isolated by a single step of adhesion to cell culture plastic, and purified via replacement of medium and a serial of passage, then the cells were identified by surface molecules CD90, CD44 and CD45 by flow cytometry. Enzyme immunocytochemistry and indirect immunofluorescence were used to evaluate the availability of ZUC3 expression by rat MSCs as a surface marker. Then further exploratory researches were carried out concerning ZUC3 expression by rat MSCs during passages (P1 to P5) and multiple differentiation (neuron, osteoblasts and adipocytes) in the certain condition. Results: Homogeneous rat MSCs could be obtained in vitro, which were uniformly positive for adhesion molecules CD90, CD44, and negative for CD45. The McAb was specifically reactive with rat MSCs as the positive cells were more than 99% by immunohistochemistry and immunofluorescence staining, and ZUC3 antigen located on the membrane of rat MSCs. The flow cytometric analysis show ZUC3 antigen expression by rat MSCs from P1 to P5 were all more than 85%. Analysis by multiple comparison, it was found some differences between P2 and P1 (93.95±2.44% v.s. 86.90±1.80%, P<0.01). The maximal expression was reached at P3 (97.10±1.25%), and the flow cytometric analysis showed a single symmetrical peak. Data of P4 (94.50±2.23%) population were slightly lower than P3 (P>0.05). By contrast, P5 (88.35±2.99%) showed a significant decline comparing with the former passages (P<0.01). Furthermore, rat MSCs could be successfully induced to differentiate into neuron-like cells, osteoblasts, and adipocytes and there was to some extent a downward trend of ZUC3 expression after differentiation (P<0.01). More than 90% rat MSCs could transform to an neuron-like appearance which were positive for NeuN, NF-M after treated with alpha-thioglycerol, and there was some downward degree of ZUC3 expression (97.77±1.03% to 80.07±2.70%, P<0.01). During the osteoblastic differentiation, it was observed an obvious down-regulation of ZUC3 expression from the 10th day (96.63±1.03% to 90.07±2.40%, P<0.01 ) and percentage on the 10th (90.07±2.40%), 15th (84.43±2.80%), 20th (64.53±7.63%) and 25th (53.40±10.02%) day were significantly lower than their anterior time respectively (P<0.05). The results of adipogenic differentiation after MSCs incubated with proper medium were similar to what observed during osteoblastic differentiation and ZUC3 expression were down-regulation on the 7th (84.33±2.70%), 14th (75.90±2.00%) and 21st (70.57±0.47%) day compared with their anterior dots respectively (P<0.01). Conclusion: ZUC3 antigen could be used for identification of rat MSCs. Significant decline of ZUC3 expression had be observed after rat MSCs were induced to differentiate along neuronal, osteoblastic and adipogenic pathways, which indicated that ZUC3 antigen would be a marker of progenitor.

scholarly journals Flow cytometric analysis of the inhibition of human basophil activation by histamine high dilutions - a replication study

2021 ◽  
Vol 11 (40) ◽  
pp. 172-173
Author(s):  
Chantal Wälchli ◽  
Stephan Baumgartner
Keyword(s):  
Flow Cytometry ◽  
Flow Cytometric Analysis ◽  
Systematic Errors ◽  
Basophil Activation ◽  
Human Basophil ◽  
Specific Marker ◽  
Flow Cytometric ◽  
High Dilutions ◽  
Independent Replication ◽  
Human Basophils

Background: Inhibition of human basophil activation by highly diluted histamine was reported to be a reliable experimental model to examine biological effects of high dilutions. However, independent replications did not always yield concordant results. Aims: We aimed at performing an independent replication of a former study [1] using rigorously controlled experimental conditions to minimise confounding factors. Materials and Methods: In 20 independent experiments, human basophils were treated with highly diluted histamine (15cH, 16cH, corresponding to 10-30-10-32 M) prior to activation by fMLP (formyl-methionyl-leucyl-phenylalanine peptide). Controls were treated with analogously diluted water (15cH, 16cH). The dilutions were prepared freshly for each experiment in deionised water by successive steps of centesimal dilution and agitation (10 s vortex at high speed). Highly diluted samples were blinded and randomised. All samples were set in triplicates. Activated basophils were determined by flow cytometry using anti-CD203c. 20 independent systematic negative control (SNC) experiments were carried out to investigate possible systematic errors. Results: No difference in basophil activation was observed between the highly diluted histamine samples and the highly diluted water controls. There was no evidence for a blood donor specificity of the results. The SNC experiments demonstrated the stability of the test system. Experimental variability within and between experiments was slightly reduced for the highly diluted histamine samples. Discussion: This study was designed as an independent reproduction of a former study [1]. Though we strictly adopted the experimental procedure described in [1], our results do not confirm the large inhibitory effects observed for histamine 15cH and 16cH. This lack of reproducibility might be due to minor differences in the experimental design, such as blinding and randomising of the samples, which we chose to perform in order to reduce the possibility of artifacts but was omitted in the former study. Conclusions: Laboratory independent replication of homeopathic basic research experiments is still a challenge. Assuming that the results formerly obtained with this model were not due to systematic errors, the quest identifying the crucial factors for successful reproducibility is open for future research. Keywords: Human basophils; histamine; high dilutions; flow cytometry Reference: [1] Sainte-Laudy J, Belon P. Improvement of flow cytometric analysis of basophil activation inhibition by high histamine dilutions. A novel basophil specific marker: CD 203c. Homeopathy. 2006;95:3-8.

Quantitative Assessment of Myeloid Nuclear Differentiation Antigen Distinguishes Myelodysplastic Syndrome From Normal Bone Marrow.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1754-1754
Author(s):  
Head R. Head ◽  
Sara A. McClintock-Treep ◽  
Leanne flye-Blakemore ◽  
Claudio Mosse ◽  
Madan Jagasia ◽  
...  
Keyword(s):  
Flow Cytometric Analysis ◽  
Bimodal Distribution ◽  
Refractory Anemia ◽  
Low Grade ◽  
Differentiation Antigen ◽  
Down Regulation ◽  
Flow Cytometric ◽  
Nuclear Differentiation ◽  
Quantitative Flow ◽  
Myeloid Progenitors

Abstract Abstract 1754 Poster Board I-780 Introduction Definitive diagnosis and classification of MDS are often difficult because of variable presence of diagnostic criteria and imprecision and ambiguities of interpretation of both morphologic and ancillary data. An objective criterion that reliably distinguishes MDS from normal marrow would greatly facilitate diagnosis, and might contribute to subclassification of MDS. Gene expression profiling has identified marked (7 fold) down-regulation of myeloid nuclear differentiation antigen (MNDA) message in cases of MDS (Hofmann WK, et al, Blood 2002;100:3553-60), and we have previously shown down-regulation of MNDA protein expression in random MDS cases using immunohistochemistry and flow cytometry (Briggs RC, et al. Cancer Research 2006;66:4645-51). To continue our previous analyses, we evaluated MNDA expression in myeloid progenitors using quantitative flow cytometric analysis in MDS and normal control marrow samples. Patients and Methods The study included 20 MDS patients receiving only supportive care undergoing bone marrow sampling for clinical purposes, and 19 frequency age-matched normal controls undergoing orthopedic surgery with no antecedent primary hematologic abnormalities. MDS diagnosis was based on 2008 WHO criteria. Quantitative flow cytometric analysis was performed with an FC500 flow cytometer (Beckman Coulter, Fullerton, CA). Cells were treated with CD45-PE and CD34-ECD (Beckman Coulter), washed in PBS with 2% FCS, and permeabilized with PermiFlow. MNDA-Alexa-488 was added at a granulocyte-monocyte specific concentration, with analysis using Winlist 5.0 software (Verity Software, Topsham, ME) with DDE links to ModFitLT 3.0 using modifications of published methods. Differentiating myeloid progenitors were identified as high side scatter/intermediate CD45/CD34-negative cells, with lymphocytes (low side scatter/high CD45/CD34-negative cells) serving as an internal dim MNDA control in each sample. Results MDS cases consisted of 12 patients with refractory cytopenia with multilineage dysplasia (RCMD), 3 with refractory anemia with excess blasts-1(RAEB-1), 4 with RAEB-2, and 1 with therapy-related MDS. In myeloid progenitors in MDS patients, the median percent of MNDA-dim cells was 67.4% (range 0.7-97.5%, interquartile range 44.9-82.7%). The analogous median percent of MNDA-dim cells in control patients was 1.2% (range 0.2-13.7%, interquartile range 0.6-2.7%). The area under the ROC curve was 0.96 (p = 9×10-7), indicating almost complete discrimination between cases and controls. 19 of 20 MDS patients demonstrated bimodal distribution of MNDA expression comprising a distinct MNDA-dim population and a separate MNDA-normal population, suggesting an admixture of MDS and normal cells. 18 of 19 control patients demonstrated a single population of MNDA-normal cells without evidence of a bimodal distribution. The single MDS patient with normal MNDA expression had prior clinical and laboratory features suggestive of refractory anemia with ringed sideroblasts. The single pediatric MDS patient had reduced MNDA expression similar to other high grade MDS samples. Patients showed trends for MDS subtype (p = 0.21) and IPSS score (p = 0.07) versus percent MNDA-dim myeloid progenitors. These data demonstrate remarkable sensitivity and specificity for use of MNDA expression to detect MDS. Conclusions Quantitative flow cytometric analysis of MNDA expression in marrow myeloid progenitors is a promising objective test for diagnosis of MDS, demonstrating a striking difference of MNDA expression in myeloid progenitors in MDS versus control patients. Our results require elaboration with analysis of low grade cases. A single possible low grade MDS case in this series demonstrated MNDA expression identical to normal control samples. Our results also require elaboration in cases constituting the differential diagnosis of MDS to evaluate the clinical utility of MNDA evaluation. The biological significance of down-regulation of MNDA in MDS is uncertain, although MNDA has been implicated in regulation of programmed cell death. Our results suggest a mix of normal and abnormal myelopoiesis in MDS patients, as predicted by cytogenetics in many cases of MDS. In summary, testing of MNDA expression in myeloid progenitors shows great promise as an objective test for diagnosis of MDS in marrow samples. Disclosures No relevant conflicts of interest to declare.

scholarly journals Porcupine and CBP/β-catenin are the most suitable targets for the inhibition of canonical Wnt signaling in colorectal carcinoma cell lines*

2020 ◽  
Vol 74 ◽  
pp. 224-235
Author(s):  
Robert Kleszcz ◽  
Violetta Krajka-Kuźniak ◽  
Jarosław Paluszczak
Keyword(s):  
Wnt Signaling ◽  
Cell Lines ◽  
Target Genes ◽  
Flow Cytometric Analysis ◽  
Molecular Targets ◽  
Canonical Wnt Signaling ◽  
Down Regulation ◽  
Flow Cytometric ◽  
Canonical Wnt ◽  
Hct116 Cells

Aim: The activation of canonical Wnt pathway is etiologically associated with the development of colorectal cancers. There are many possible molecular targets for the therapeutic abrogation of Wnt/β-catenin signaling. The aim of this study was to select the best molecular targets for the attenuation of β-catenin-dependent gene expression in colorectal cancer cell lines. Material/Methods: An siRNA screen was used for the selection of the best molecular targets for the down-regulation of TCF/LEF-dependent GFP expression in HCT116 cells. The level of the expression of β-catenin target genes was analyzed by qPCR. The effect of the tested chemicals on cell migration, cell cycle and apoptosis was assessed by the wound healing assay, flow cytometric analysis of propidium iodide stained cells and flow cytometric analysis of the activity of caspases-3/7, respectively. Results: Of the forty three genes which were tested in the screening stage, eight (KDM6A, KDM1A, PORCN, KDM4C, CARM1, DVL1, CBP, KMT2A) were selected as most promising. Small molecule inhibitors of these proteins (GSK-J4, GSK-LSD1, IWP-2, ML324, MS049, Dvl-PDZ Domain Inhibitor II, PRI-724, MM-102) were further used. The inhibitors of Porcupine (IWP-2) and CBP (PRI-724) were most effective in the down-regulation of the expression of β-catenin target genes and the induction of apoptosis in HCT116 cells, but showed weaker effects in DLD-1 cells. Conclusions: The inhibition of CBP and Porcupine is the most effective in attenuating canonical Wnt signaling in colon cancer cells. Future studies should determine which factors affect the sensitivity towards these promising anti-cancer agents.

Apoptotic Effects of Melittin on 4T1 Breast Cancer Cell Line is associated with Up Regulation of Mfn1 and Drp1 mRNA Expression

2020 ◽  
Vol 20 (7) ◽  
pp. 790-799 ◽  
Author(s):  
Farnaz D. Moghaddam ◽  
Pejman Mortazavi ◽  
Somayeh Hamedi ◽  
Mohammad Nabiuni ◽  
Nasim H. Roodbari
Keyword(s):  
Breast Cancer ◽  
Mrna Expression ◽  
Hemolytic Activity ◽  
Breast Cancer Cells ◽  
Flow Cytometric Analysis ◽  
Control Group ◽  
Flow Cytometric ◽  
4T1 Cells ◽  
4T1 Breast Cancer ◽  
Apoptotic Effects

Background and Purpose: Melittin, as the main ingredient of honeybee venom, that has shown anticancer properties. The present study aimed at investigating the cytotoxic impacts of melittin on 4T1 breast cancer cells. Methods: Hemolytic activity of different concentrations (0.125, 0.25, 0.5, 1, 2, 4, 8μg/ml) of melittin was assayed and then cytotoxicity of selected concentrations of melittin (2, 4, 8, 16, 32, and 64μg/ml), 2 and 4μg/ml of cisplatin and 0.513, 0.295 and 0.123μg/ml of doxorubicin was evaluated on 4T1 cells using MTT assay. We used Morphological evaluation and flow cytometric analysis was used. Real time PCR was also used to determine mRNA expression of Mfn1 and Drp1 genes. Results: All compounds showed anti-proliferative effects on the tumor cell line with different potencies. Melittin had higher cytotoxicity against 4T1 breast cancer cells (IC50= 32μg/ml-72h) and higher hemolytic activity (HD50= 1μg/ml), as compared to cisplatin and doxorubicin. Mellitin at 16 and 32μg/ml showed apoptotic effects on 4T1 cells according to the flow cytometric analysis. The Real time PCR analysis of Drp1 and Mfn1 expression in cells treated with 16μg/ml of melittin revealed an up-regulation in Drp1 and Mfn1 genes mRNA expression in comparison with control group. Treatment with 32μg/ml of melittin was also associated with a rise in mRNA expression of Drp1 and Mfn1 as compared to the control group. Conclusion: The results of this study showed that melittin has anticancer effects on 4T1 cell lines in a dose and time dependent manner and can be a good candidate for further research on breast cancer treatment.

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