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Peer-reviewed

A Step-by-step Protocol for Obtaining Mature Microglia from Mice

从小鼠中获取成熟小胶质细胞的方法

MY Min-Jung Yoo
MK Min-Soo Kwon

发布: 2022年08月05日第12卷第15期 DOI: 10.21769/BioProtoc.4481 浏览次数: 2299

评审: Alessandro DidonnaYiqun YuThirupugal Govindarajan

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Cover of Journal of Neuroinflammation, featuring study using the protocol.

Dec 2021

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Abstract

In mice, microglial precursors in the yolk sac migrate to the brain parenchyma through the head neuroepithelial layer between embryonic days 8.5 (E8.5)–E16.5 and acquire their unique identity with a ramified form. Based on the microglial developmental process, we dissected the neuroepithelial layer (NEL) of E13.5 mice, which is composed of microglial progenitor and neuroepithelial cells. The NEL was bankable and expandable. In addition, microglial precursors were matured according to NEL culture duration. The matured microglia (MG; CD11b-positive cells) were easily isolated from the cultured NEL using a magnetic-activated cell sorting system and named NEL-MG. In conclusion, we obtained higher yields of adult-like microglia (mature microglia: NEL-MG) compared to previous in vitro surrogates such as neonatal microglia and microglial cell lines.


Graphical abstract:




Keywords: Microglia (小胶质细胞) search Neuroepithelial layer (NEL) (神经上皮层 (NEL)) search Microglial progenitor (小胶质细胞祖细胞) search Expandable cells (可扩展的细胞) search Banking (储存) search

Background

So far, microglial cell lines, primary fetal/neonatal microglia, and acute isolated adult microglia have been used in in vitro studies. However, low yields, immature phenotypes, and the use of many experimental animals are barriers to studying microglia. Here, we introduce a new method for obtaining bankable and expandable adult-like microglia. The neuroepithelial layer (NEL) of mice at embryonic day 13.5 (E13.5), which is composed of microglial progenitors and neuroepithelial cells, was dissected and then cultured or banked. Microglia (MG; CD11b-positive cells) were isolated from the cultured NEL using a magnetic-activated cell sorting system and named NEL-MG. This new method contributes to the obtainment of matured forms of microglia (adult-like microglia) with only a small number of experimental animals.

Materials and Reagents

Animals

Timed pregnant (13d; TP13) C57BL/6 mice (female, Daihan-Biolink Co., Chungbuk, Korea)


Culture products

  1. Microtubes (Axygen, catalog number: MCT-150-C)

  2. 15 mL conical tubes (Thermo Fisher Scientific, catalog number: 339650)

  3. 100 mm dish (Thermo Fisher Scientific, catalog number: 150466)

  4. T-25 Flasks (Thermo Fisher Scientific, catalog number: 156367)

  5. Hanks’ balanced salt solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14170-112)

  6. 75% ethyl alcohol anhydrous (Daejung Chemicals & Metals, catalog number: 4023-2304)

  7. Trypsin 2.5% (Thermo Fisher Scientific, GibcoTM, catalog number: 15090-046)

  8. Poly-D-lysine (PDL) (Sigma-Aldrich, catalog number: P7280-5MG)

  9. Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11995-065)

  10. Penicillin-Streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122)

  11. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 16000-044)

  12. GlutaMAXTM (Thermo Fisher Scientific, GibcoTM, catalog number: 35050061)

  13. Dulbecco’s phosphate buffered salt (DPBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-144)

  14. Trypan blue stain (0.4%) (Thermo Fisher Scientific, GibcoTM, catalog number: 15250-061)

  15. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650-5X5ML)

  16. Bovine serum albumin fraction V (BSA) (Merck, catalog number: 10735086001)

  17. CD11b (microglia) MicroBeads (Miltenyi Biotec, catalog number: 130-093-634)

  18. MS columns (Miltenyi Biotec, catalog number: 130-042-201)

  19. Culture medium (500 mL) (see Recipes)

  20. 70% ethanol (100 mL) (see Recipes)

  21. 0.25% trypsin (10 mL) (see Recipes)

  22. PB buffer (50 mL) (see Recipes)

Equipment

  1. Optical microscope (Olympus, model: SZ-ST)

  2. Clean bench (LabTech, model: LCB-1201V)

  3. Dissection tools

    1. Mosquito forceps (KASCO, catalog number: S8-099)

    2. Micro dissecting forceps (KASCO, catalog number: 50-2000-1)

    3. Micro scissors (Medro Instruments, catalog number: 02-027-10)

    4. Electronic forceps (KASCO, catalog number: 11-412-11)

  4. Centrifuge machine (LaboGene, catalog number: 1248R)

  5. Hemocytometer (Marienfeld superior, catalog number: HSU-0650030)

  6. CO2 cell culture incubator (PHCbi, catalog number: MCO-18AC-PK)

  7. 37 °C water bath (DAIHAN Scientific, catalog number: DH.WHB00106)

  8. Magnetic cell separator (Miltenyi Biotec, catalog number:130-042-102)

Software

  1. ImageJ (National Institutes of Health, https://imagej.net)

  2. Prism 7 (GraphPad, https://graphpad.com)

  3. Biorender (Biorender, https://biorender.com)

Procedure

English
中文翻译

文章信息

版权信息

© 2022 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

You, M. J. and Kwon, M. S. (2022). A Step-by-step Protocol for Obtaining Mature Microglia from Mice. Bio-protocol 12(15): e4481. DOI: 10.21769/BioProtoc.4481.

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分类

神经科学 > 神经系统疾病

细胞生物学 > 细胞分离和培养 > 细胞分化

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