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Peer-reviewed

Isolation of CD31+ Bone Marrow Endothelial Cells (BMECs) from Mice

从小鼠中分离 CD31+ 骨髓内皮细胞 (BMECs)

AS Alhaji Osman Smith
SA Seyram Yao Adzraku
WJ Wen Ju
JQ Jianlin Qiao
KX Kailin Xu
LZ Lingyu Zeng

发布: 2021年11月20日第11卷第22期 DOI: 10.21769/BioProtoc.4227 浏览次数: 3031

评审: Alessandro DidonnaJungeun YuAnonymous reviewer(s)

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Cover of Stem Cell Research & Therapy, featuring study using the protocol.

May 2021

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Abstract

In the bone marrow microenvironment, endothelial cells (ECs) play a pivotal role in regulating the production of both growth and inhibiting factors. They are held together by adherence molecules that interact with hematopoietic progenitor cells. The study of ECs in the hematopoietic stem cell niche is limited due to the lack of efficient protocols for isolation. In this protocol, we developed a two-step approach to extract bone marrow endothelial cells (BMECs) to unlock the challenges researchers face in understanding the function of the endothelial vascular niche in in-vitro studies.

Keywords: Bone marrow endothelial cells (BMECs) (骨髓内皮细胞(BMECs)) search Endothelial cells (ECs) (内皮细胞(ECs)) search Extraction (抽取) search Mouse bone marrow (小鼠骨髓) search Isolation (分离) search Endothelial-vascular niche (内皮血管微环境) search

Background

The bone marrow microenvironment (BMM) is the compilation of cells and vascular networks within the marrow that acts as a vital and dynamic support system to assist hematopoiesis. The cellular component of the bone marrow consists of hematopoietic and non-hematopoietic cells, such as endothelial cells (ECs), osteoblasts, osteoclasts, adipocytes, and fibroblasts (Mishra et al., 2011; Birbrair and Frenette, 2016). The presence of ECs within the bone marrow environment regulates the trafficking and homing of the hematopoietic progenitor and stem cells. Alteration of the bone marrow endothelial cells (BMECs) due to malignant cells causes activation of endothelial-derived signaling pathways that favor the proliferation, differentiation, migration, and survival of neoplastic cells. It also stimulates angiogenesis, disrupting homeostasis within the BMM (Coşkun and Hirschi, 2015; Isern and Mendez-Ferrer, 2011; Passweg et al., 2016). In this protocol, we describe the isolation of BMECs and characterize their morphology.

Materials and Reagents

  1. 1.5 ml Eppendorf centrifuge tube (Eppendorf, catalog number: NA)

  2. 0.5 ml Nest micro-centrifuge tube (Biosharp, Life Science, catalog number: 201214)

  3. 18-gauge needle and syringe (Lujie®, 2015315175)

  4. Sterile 15 ml and 50 ml conical tubes (Beijing Labgic Technology, catalog numbers: CT-002-15 and CT-002-50)

  5. 100 mm cell culture dish (Thermo Fisher Scientific) and 12-48 well plates (Costar®, Corning, catalog number: 430161)

  6. Micro-dissecting board and pins

  7. Sterile gloves and gauze

  8. 70 µm cell strainer (Biologix, catalog number: 15-1070)

  9. Pipette calibrators P1000 (N253062), P200 (MZ26504), and P10 (N252362)

  10. Magnetic separation column and MACS® Separator (MACS, Miltenyi Biotec)

  11. Wild-type mice (C57BL/6)

    Male and female wild-type mice aged 8-12 weeks old were obtained from Jackson laboratory. Mice were housed under the regulation of Xuzhou Medical University, Jiangsu Province, China.

  12. 70-80% ethanol

  13. Milli-Q water (Quantum® Tix, catalog number: QTUMOTIX1)

  14. Miltenyi Biotec CD 31 microbeads, MS column (miniMACSTM Separator, Miltenyi Biotec)

  15. Fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific)

  16. Bovine serum albumin (BSA) (Solarbio® Life Science)

  17. Distilled water

  18. Accutase solution (Absin, Biochemical Company/ Thermo Fisher Scientific)

  19. Hydrochloride acid (HCl)

  20. Potassium chloride (KCl)

  21. Sodium chloride (NaCl)

  22. Sodium phosphate Dibasic (Na2HPO4)

  23. Potassium phosphate monobasic (KH2PO4)

  24. 10,000 U/ml penicillin /streptomycin (Gibco, Thermo Fisher Scientific)

  25. Rat-tail collagen type-I 3 mg/ml (Thermo Fisher Scientific, catalog number: A10483-01)

  26. Human plasma fibronectin purified protein (EMD Millipore, catalog number: 3383980 or FC010)

  27. PECAM-1 (CD31), rabbit-polyclonal (Affinity Bioscience, catalog number: AF6191)

  28. VE-CADHERIN (CD144) rabbit-polyclonal (Affinity Bioscience, catalog number: AF6265)

  29. Vascular endothelial growth factor receptor-2 (VEGFR2 (FlK-1) rabbit-polyclonal (Bioworld Technology, catalog number: Q1169)

  30. Secondary antibodies: Goat Anti-rabbit (IgG) Alexa Fluor® 594 (green; Abcam, catalog number: Ab15008) and Alexa Fluor®488 (red; Abcam, catalog number: Ab15007)

  31. Endothelial cells medium (EBM-2) (Lonza, Clonetics®)

  32. 1× Dulbecco’s phosphate buffer solution (DPBS) (see Recipes)

  33. Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Thermo Fisher Scientific) (see Recipes)

  34. 0.5 M Disodium Ethylenediaminetetraacetic acid (EDTA)·2H2O (see Recipes)

  35. 0.001 M Ethylenediaminetetraacetic acid (EDTA)/DBPS (see Recipes)

  36. Sterile harvesting buffer solution (see Recipes)

  37. Complete endothelial cell medium (see Recipes)

  38. 4% Paraformaldehyde (PFA) solution (Sinopharm Chemical Reagent) (see Recipes)

  39. 1% bovine serum albumin (BSA/DPBS) (see Recipes)

  40. 0.5 M EDTA (see Recipes)

  41. Dish pre-coating reagents (see Recipes)

Equipment

  1. Surgical scissors/forceps (10-11 cm long stainless steel dissecting scissors and 10-11 cm long stainless steel dissecting straight forceps)

  2. Cell culture incubator (CO2 incubator) (Thermo Fisher Scientific, model: Heracell 150i, catalog number: 41629032)

  3. Pipettes: P10, P200, and P1000 (Thermo Fisher Scientific, catalog numbers: 464000, 4640050, 4640060)

  4. Cell counting chamber (hemocytometer)/automated cell counting machine (Mindray)

  5. Refrigerator (2-8°C) (Haier, Bio-Medical)

  6. -20°C freezer (Haier)

  7. Laminar flow hook (Thermo Fisher Scientific, SN-194564, model -1381)

  8. High-speed centrifuge (Beckman Coulter, model: Microfuge®20R, catalog number: MRZ14H028, and low-speed centrifuge AIRTECH-KDC-40 Anhui USTC Zonkia Scientific instrument co.Ltd, catalog number: 02241700049)

  9. Nikon Eclipse microscope (Nikon, model: Eclipse Ti)

  10. Sterilization machine (Hirayama, model: HICLAVETM HVE-50, catalog number: 30613065826)

  11. pH meter and automated weighing machine (Metter Toledo)

  12. Magnetic stirrer (IKA® RCT Basic, 0317090005661)

  13. Graduate cylinder

Software

  1. Flow cytometry software

  2. FlowJo software version 7.6.2

Procedure

English
中文翻译

文章信息

版权信息

© 2021 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Smith, A. O., Adzraku, S. Y., Ju, W., Qiao, J., Xu, K. and Zeng, L. (2021). Isolation of CD31+ Bone Marrow Endothelial Cells (BMECs) from Mice. Bio-protocol 11(22): e4227. DOI: 10.21769/BioProtoc.4227.

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分类

干细胞 > 成体干细胞 > 内皮干细胞/祖细胞

细胞生物学 > 细胞分离和培养 > 细胞分离

细胞生物学 > 细胞分离和培养 > 单层培养

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