Isolation and Flow Cytometric Analysis of the Stromal Vascular Fraction Isolated from Mouse Adipose Tissue

2018 ◽  
pp. 1-9
Author(s):  
Annie C. Bowles ◽  
Alan Tucker ◽  
Bruce A. Bunnell
Keyword(s):  
Adipose Tissue ◽  
Flow Cytometric Analysis ◽  
Stromal Vascular Fraction ◽  
Flow Cytometric

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.

scholarly journals Gpr1 is an active chemerin receptor influencing glucose homeostasis in obese mice

2014 ◽  
Vol 222 (2) ◽  
pp. 201-215 ◽  
Author(s):  
Jillian L Rourke ◽  
Shanmugam Muruganandan ◽  
Helen J Dranse ◽  
Nichole M McMullen ◽  
Christopher J Sinal
Keyword(s):  
Adipose Tissue ◽  
Glucose Homeostasis ◽  
Stromal Vascular Fraction ◽  
Tolerance Test ◽  
Glucose Levels ◽  
Brown Adipose ◽  
Elevated Glucose ◽  
And Function ◽  
G Protein Coupled

Chemerin is an adipose-derived signaling protein (adipokine) that regulates adipocyte differentiation and function, immune function, metabolism, and glucose homeostasis through activation of chemokine-like receptor 1 (CMKLR1). A second chemerin receptor, G protein-coupled receptor 1 (GPR1) in mammals, binds chemerin with an affinity similar to CMKLR1; however, the function of GPR1 in mammals is essentially unknown. Herein, we report that expression of murineGpr1mRNA is high in brown adipose tissue and white adipose tissue (WAT) and skeletal muscle. In contrast to chemerin (Rarres2) andCmklr1,Gpr1expression predominates in the non-adipocyte stromal vascular fraction of WAT. Heterozygous and homozygousGpr1-knockout mice fed on a high-fat diet developed more severe glucose intolerance than WT mice despite having no difference in body weight, adiposity, or energy expenditure. Moreover, mice lackingGpr1exhibited reduced glucose-stimulated insulin levels and elevated glucose levels in a pyruvate tolerance test. This study is the first, to our knowledge, to report the effects ofGpr1deficiency on adiposity, energy balance, and glucose homeostasisin vivo. Moreover, these novel results demonstrate that GPR1 is an active chemerin receptor that contributes to the regulation of glucose homeostasis during obesity.

scholarly journals Long-Term Severe In Vitro Hypoxia Exposure Enhances the Vascularization Potential of Human Adipose Tissue-Derived Stromal Vascular Fraction Cell Engineered Tissues

2021 ◽  
Vol 22 (15) ◽  
pp. 7920
Author(s):  
Myroslava Mytsyk ◽  
Giulia Cerino ◽  
Gregory Reid ◽  
Laia Gili Sole ◽  
Friedrich S. Eckstein ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Therapeutic Potential ◽  
Stromal Vascular Fraction ◽  
Human Adipose Tissue ◽  
Bioreactor Culture ◽  
Proliferating Cells ◽  
Angiogenic Potential ◽  
Engineered Tissues

The therapeutic potential of mesenchymal stromal/stem cells (MSC) for treating cardiac ischemia strongly depends on their paracrine-mediated effects and their engraftment capacity in a hostile environment such as the infarcted myocardium. Adipose tissue-derived stromal vascular fraction (SVF) cells are a mixed population composed mainly of MSC and vascular cells, well known for their high angiogenic potential. A previous study showed that the angiogenic potential of SVF cells was further increased following their in vitro organization in an engineered tissue (patch) after perfusion-based bioreactor culture. This study aimed to investigate the possible changes in the cellular SVF composition, in vivo angiogenic potential, as well as engraftment capability upon in vitro culture in harsh hypoxia conditions. This mimics the possible delayed vascularization of the patch upon implantation in a low perfused myocardium. To this purpose, human SVF cells were seeded on a collagen sponge, cultured for 5 days in a perfusion-based bioreactor under normoxia or hypoxia (21% and <1% of oxygen tension, respectively) and subcutaneously implanted in nude rats for 3 and 28 days. Compared to ambient condition culture, hypoxic tension did not alter the SVF composition in vitro, showing similar numbers of MSC as well as endothelial and mural cells. Nevertheless, in vitro hypoxic culture significantly increased the release of vascular endothelial growth factor (p < 0.001) and the number of proliferating cells (p < 0.00001). Moreover, compared to ambient oxygen culture, exposure to hypoxia significantly enhanced the vessel length density in the engineered tissues following 28 days of implantation. The number of human cells and human proliferating cells in hypoxia-cultured constructs was also significantly increased after 3 and 28 days in vivo, compared to normoxia. These findings show that a possible in vivo delay in oxygen supply might not impair the vascularization potential of SVF- patches, which qualifies them for evaluation in a myocardial ischemia model.

scholarly journals Flow cytometric analysis of T lymphocytes and cytokines in aqueous humor of patients with varicella zoster virus-mediated acute retinal necrosis

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Hao Kang ◽  
Yunbo Wei ◽  
Ming Liu ◽  
Di Yu ◽  
Yong Tao
Keyword(s):  
T Lymphocytes ◽  
Varicella Zoster Virus ◽  
Aqueous Humor ◽  
T Lymphocyte ◽  
Lymphocyte Subsets ◽  
Flow Cytometric Analysis ◽  
T Lymphocyte Subsets ◽  
Varicella Zoster ◽  
Flow Cytometric ◽  
Cd8 T Lymphocytes

Abstract Background The purpose of this study is to investigate the aqueous humor (AH) T lymphocyte subsets and cytokines of acute retinal necrosis (ARN) to elucidate the immunologic inflammatory features of this disorder. Methods Three patients with ARN infected with varicella zoster virus (VZV) who underwent multiple intravitreal injections of ganciclovir were enrolled in this study. The control group consisted of four non-infectious patients with acute anterior uveitis (AAU). Flow cytometric analysis was performed on the lymphocyte subsets from the AH and peripheral blood (PB) samples during the active phase of intraocular inflammation. Five inflammatory cytokines were measured in each AH sample and various clinical characteristics were also assessed. Results VZV deoxyribonucleic acid (DNA) was detected by real-time polymerase chain reaction (PCR) in AH from all the ARN patients, who showed higher CD8+ T lymphocytes population in AH than the AAU patients (p = 0.006). CD4/CD8 ratios of T lymphocytes and the percentage of CD8 + CD25+ T lymphocytes in AH were significantly lower in ARN than in AAU (p = 0.006; p = 0.012). In the ARN patients, the percentages of CD4+ and CD8+ T lymphocytes in AH were higher than those found in PB. The percentage of CD4 + CD25+ T lymphocytes in AH was significantly higher than the proportion in PB in the AAU patients (p = 0.001). Immunoregulatory cytokine Interleukin-10 in AH was significantly elevated in the ARN patients in comparison with the case of the AAU patients (p = 0.036). In ARN, the copy number of VZV DNA in AH positively correlated with the percentage of CD8+ T lymphocytes in AH and negatively correlated with the CD4/CD8 ratio in AH during the course of disease treatment (p = 0.009, r = 0.92; p = 0.039, r = − 0.834). Conclusion The ARN patients caused by VZV had different intraocular T lymphocyte subsets and cytokines profile than those of the non-infectious patients. High percentages of CD8+ T lymphocytes and low CD4/CD8 T cell ratios may be a potential biomarker for diagnosis of viral-infectious uveitis. T lymphocytes examination at the inflammatory sites has the potential to become a useful research tool for differentiating viral and non-viral uveitis.

scholarly journals Derivation, characterization, and in vitro cell regeneration of canine white adipose tissue-derived mesenchymal stem cells obtained from a mesenteric region

2021 ◽  
Vol 82 (1) ◽  
Author(s):  
Anirban Mandal ◽  
Ajeet Kumar Jha ◽  
Dew Biswas ◽  
Shyamal Kanti Guha
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
White Adipose Tissue ◽  
Stromal Vascular Fraction ◽  
Cell Regeneration ◽  
Wound Healing Assay ◽  
Chain Reaction ◽  
Polymerase Chain

Abstract Background The study was conducted to assess the characterization, differentiation, and in vitro cell regeneration potential of canine mesenteric white adipose tissue-derived mesenchymal stem cells (AD-MSCs). The tissue was harvested through surgical incision and digested with collagenase to obtain a stromal vascular fraction. Mesenchymal stem cells isolated from the stromal vascular fraction were characterized through flow cytometry and reverse transcription-polymerase chain reaction. Assessment of cell viability, in vitro cell regeneration, and cell senescence were carried out through MTT assay, wound healing assay, and β-galactosidase assay, respectively. To ascertain the trilineage differentiation potential, MSCs were stained with alizarin red for osteocytes, alcian blue for chondrocytes, and oil o red for adipocytes. In addition, differentiated cells were characterized through a reverse transcription-polymerase chain reaction. Results We observed the elongated, spindle-shaped, and fibroblast-like appearance of cells after 72 h of initial culture. Flow cytometry results showed positive expression for CD44, CD90, and negative expression for CD45 surface markers. Population doubling time was found 18–24 h for up to the fourth passage and 30±0.5 h for the fifth passage. A wound-healing assay was used to determine cell migration rate which was found 136.9 ± 4.7 μm/h. We observed long-term in vitro cell proliferation resulted in MSC senescence. Furthermore, we also found that the isolated cells were capable of differentiating into osteogenic, chondrogenic, and adipogenic lineages. Conclusions Mesenteric white adipose tissue was found to be a potential source for isolation, characterization, and differentiation of MSCs. This study might be helpful for resolving the problems regarding the paucity of information concerning the basic biology of stem cells. The large-scale use of AD-MSCs might be a remedial measure in regenerative medicine.

scholarly journals LncRNA NEAT1 Enhances Glioma Progression via Regulating the miR-128-3p/ITGA5 Axis

2021 ◽  
Author(s):  
Jiakai Chen ◽  
Handong Wang ◽  
Junjun Wang ◽  
Wenhao Niu ◽  
Chulei Deng ◽  
...  
Keyword(s):  
Flow Cytometric Analysis ◽  
Biological Functions ◽  
Quantitative Real Time Pcr ◽  
Flow Cytometric ◽  
Non Coding Rna ◽  
Rna Immunoprecipitation ◽  
Human Gbm ◽  
Novel Strategy ◽  
Lncrna Neat1 ◽  
Long Non Coding Rna

AbstractAccumulating evidences indicate that long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) promotes the progression of glioma. In this study, we postulated that NEAT1 may act as a miR-128-3p sponge. Relative levels of NEAT1 and miR-128-3p expression in human glioma samples and GBM cells were detected using quantitative real-time PCR. By means of CCK-8 assays, transwell assays, and flow cytometric analysis, the biological functions of miR-128-3p and NEAT1 were investigated in U87MG and U251MG human GBM cell lines with stable miR-128-3p and NEAT1 knockdown or overexpression. The luciferase reports, RNA pull-down assay, and RNA immunoprecipitation assay were conducted to determine the relevance of NEAT1 and miR-128-3p in glioma. As a result, high expression of NEAT1 and lack of miR-128-3p were observed in glioma specimens and cells. By binding to anti-oncogene miR-128-3p in the nucleus, NEAT1 enhanced tumorigenesis and glioma development. Further experiments suggested that ITGA5 expression was increased in glioma tissues and was found to be connected with miR-128-3p. Additionally, NEAT1 facilitated ITGA5 expression via competitively binding to miR-128-3p. For this reason, ITGA5 would not be decomposed by miR-128-3p and could activate FAK signaling pathway, thereby promoting cell growth. Collectively, these results indicated that the NEAT1/miR-128-3p/ITGA5 axis was involved in glioma initiation and progression, and might offer a potential novel strategy for treatment of glioma.

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