Flow Cytometry of Neural Cells

An optimized method to process mouse CNS to simultaneously analyze neural cells and leukocytes by flow cytometry

Journal of Neuroscience Methods ◽

10.1016/j.jneumeth.2015.03.021 ◽

2015 ◽

Vol 247 ◽

pp. 23-31 ◽

Cited By ~ 24

Author(s):

Laurine Legroux ◽

Camille L. Pittet ◽

Diane Beauseigle ◽

Gabrielle Deblois ◽

Alexandre Prat ◽

...

Keyword(s):

Flow Cytometry ◽

Neural Cells

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Meningeal Whole Mount Preparation and Characterization of Neural Cells by Flow Cytometry

Current Protocols in Immunology ◽

10.1002/cpim.50 ◽

2018 ◽

Vol 121 (1) ◽

Cited By ~ 8

Author(s):

Antoine Louveau ◽

Anthony J. Filiano ◽

Jonathan Kipnis

Keyword(s):

Flow Cytometry ◽

Neural Cells ◽

Whole Mount

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Thrombopoietin Protects Neural Cells and Endothelial Cells from Apoptosis Via PI3K/AKT Pathway

Blood ◽

10.1182/blood-2020-140262 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 8-9

Author(s):

Liang Li ◽

Junyan Wang ◽

Jieyu Ye ◽

Liuming Yang ◽

Beng H Chong ◽

...

Keyword(s):

Flow Cytometry ◽

Endothelial Cells ◽

Protective Effect ◽

Cell Viability ◽

Caspase 3 ◽

Conflicts Of Interest ◽

Umbilical Vein ◽

Akt Pathway ◽

Neural Cells ◽

Pre Treatment

Background: Thrombopoietin (TPO) is a hematopoietic growth factor that regulates the production of platelets and stimulates production and differentiation. The expression of TPO and TPO receptor (c-mpl) in the central nervous system (CNS) has been identified. However, the role of TPO in neural cells and endothelial cells were not clear. Methods: C17.2 and human umbilical vein endothelial (HUVEC) cells were treated with CoCl2, TPO, or TPO + CoCl2. TPO was added into the culture medium 48 h before CoCl2 treatment. The cell viability and apoptosis of each group were tested by Cell Counter Kit 8 (CCK-8) assay and flow cytometry. The expression of Caspase-3 and mitochondrial membrane potential (MMP) were then determined by flow cytometry with Caspase-3-PE and JC-1. The effect of TPO in the PI3K/AKT pathway was detected by using Western blot. Results: TPO has a dose-dependent effect on the growth of C17.2 cells. LY-294002 pretreatment suppressed the TPO-induced AKT activation and abolished the prosurvival effect of TPO. Via the Bcl-2/BAX signaling pathway, TPO exerted an anti-apoptotic effect by suppressing mitochondria membrane potentials. We also investigated the protective effect of TPO on human endothelial cells. The cell viability of HUVECs decreased gradually with the enhancement of CoCl2 at a gradient of chemical concentrations (r= -0.997). CoCl2 dramatically increased apoptosis of HUVECs, whereas pre-treatment with TPO rescued cell apoptosis induced by CoCl2 (P<0.01). Further investigation found that TPO decreased the expression of Caspase-3 and inhibited the reduction of MMP induced by CoCl2 (P<0.05). TPO increased the activation of PI3K/AKT pathway in HUVECs. Conclusion: TPO has a protective effect against apoptosis of neural cells and endothelial cells through activating the PI3K/AKT pathway, thus decreasing the expression of apoptosis protease Caspase-3 and inhibiting the reduction of MMP. Disclosures No relevant conflicts of interest to declare.

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Abstract 15183: Thrombopoietin Has a Protective Effect Against Apoptosis of Endothelial Cells Through Activating Pi3k/akt Pathway

Circulation ◽

10.1161/circ.142.suppl_3.15183 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Liang Li ◽

Junyan Wang ◽

kuan zeng ◽

hua zhang ◽

Yanqi Yang ◽

...

Keyword(s):

Flow Cytometry ◽

Endothelial Cells ◽

Protective Effect ◽

Cell Viability ◽

Signaling Pathway ◽

Caspase 3 ◽

Umbilical Vein ◽

Neonatal Rat ◽

Akt Pathway ◽

Neural Cells

Introduction: Thrombopoietin (TPO) is a hematopoietic growth factor for platelet lineage. TPO was found to be neuroprotective in hypoxic-ischemic neonatal rat brain models and treatment with TPO reduced brain damage and improved sensorimotor functions (Li L et al, Aging-US, 2020). However, the underlying mechanism of TPO in this model, and its role in neural cells and endothelial cells were still unclear. Methods: C17.2 cells were divided into control (0.5% FCS), Normal (10% FCS), TPO and TPO + LY294002 groups. Human umbilical vein endothelial (HUVEC) cells were divided in to normal, CoCl 2 , TPO and TPO + CoCl2 groups. The expressed of TPO and c-mpl was tested by RT-PCR. The cell viability and apoptosis of each group were tested by Cell Counter Kit 8 (CCK-8) assay and flow cytometry. The expression of Caspase-3 and mitochondrial membrane potential (MMP) were then determined by flow cytometry with Caspase-3-PE and JC-1. The effect of TPO in PI3K/AKT pathway was detected by using Western blot. Results: Both TPO and c-mpl are expressed in the neurons of the human CNS. TPO was also detected in human cerebrospinal fluid. TPO promoted C17.2 cell proliferation through activation of the PI3K/Akt signaling pathway. Via the Bcl-2/BAX signaling pathway, TPO exerted an anti-apoptotic effect by suppressing mitochondria membrane potentials. We also investigated the protective effect of TPO on human endothelial cells. CoCl 2 significantly inhibited the growth of HUVECs. The cell viability of HUVECs decreased gradually with the enhancement of CoCl2 at a gradient of chemical concentrations (r= -0.997). CoCl 2 dramatically increased apoptosis of HUVECs, whereas pre-treatment with TPO rescued cell apoptosis induced by CoCl 2 (P<0.001). Further investigation found that TPO decreased the expression of Caspase-3 and inhibited the reduction of MMP induced by CoCl 2 (P<0.05). TPO increased the activation of PI3K/AKT pathway in HUVECs. Conclusions: TPO has a protective effect against apoptosis of neural cells and endothelial cells through activating the PI3K/AKT pathway, thus decreasing the expression of apoptosis protease Caspase-3 and inhibiting the reduction of MMP.

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