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Immune Cell Isolation from Mouse Femur Bone Marrow
从小鼠股骨骨髓中分离免疫细胞   

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Abstract

The bone marrow is the site of hematopoiesis and contains mixed population of blood cells including erythrocytes, granulocytes, monocytes, dendritic cells, lymphocytes and hematopoietic stem cells. The following protocol provides a simple and fast method for isolation of bone marrow immune cells (no erythrocytes) from mouse femurs with a yield of approximate 8 x 107 cells in 5 ml culture media (1.6 x 104 cells/µl). Further isolation or flow cytometric analysis might be required for study of specific immune cell types.

Keywords: Blood(血), Flow cytometry(流cytometry), Hematopoietic stem cells(造血干细胞)

Materials and Reagents

  1. Sterile paper towel
  2. Sterile surgical pad (Direct Resource, catalog number: 19015742 )
  3. 23-gauge (or 25-/26-gauge) needle (BD Biosciences, catalog number: 305145 )
  4. 10 ml syringe (BD Biosciences, catalog number: 309604 )
  5. 70 µm nylon cell strainer (Falcon, catalog number: 352350 )
    Note: Currently, it is “Corning, Falcon®, catalog number: 11995-065 ”.
  6. 50 ml conical tube (Falcon, catalog number: 21008-940 )
    Note: Currently, it is “Corning, Falcon®, catalog number: 21008-940 ”.
  7. 5 ml syringe plunger (BD Biosciences, catalog number: 309646 )
  8. Adult mice (> 6 weeks, any strain) (e.g., C57BL/6)
  9. Hank’s balanced salt solution (HBSS), no Calcium, no Magnesium, no phenol red (Life Technologies, catalog number: 14175095 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 14175095 ”.
  10. DMEM medium, high glucose, pyruvate, L-glutamine (Life Technologies, catalog number: 11995-065 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 11995-065 ”.
  11. 70% ethanol
  12. Fetal bovine serum heat inactivated (FBS) (Sigma-Aldrich, catalog number: F9665 )
  13. Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: 213330 )
  14. Potassium bicarbonate (KHCO3) (Sigma-Aldrich, catalog number: 237205 )
  15. Disodium edetate (Sigma-Aldrich, catalog number: D2900000 )
  16. RBC lysis buffer (see Recipes)
  17. DMEM medium (see Recipes)

Equipment

  1. Blunt-end sterile scissors (Thermo Fisher Scientific, Fisher Scientific, catalog number: 08-950 )
  2. Sharp sterile scissors (Thermo Fisher Scientific, Fisher Scientific, catalog number: 08-940 )
  3. Sterile forceps (Thermo Fisher Scientific, Fisher Scientific, catalog number: 08-890 )
  4. HausserTM LevyTM Hemacytometer Chamber Set (Thermo Fisher Scientific, Fisher Scientific, catalog number: 02-671-55A ) or coulter Z2 cell and particle counter (Beckman Coulter, catalog number: 383550 )
  5. Refrigerated centrifuge
  6. Sterile culture hood
  7. CO2 rodent euthanasia chamber

Procedure

  1. Euthanize the mouse with CO2 and place mouse onto a sterile surgical pad in a sterile hood. Sterilize the mouse abdomen area and skin of hindlimbs with 70% ethanol (Figure 1).


    Figure 1. Sterilization of mouse abdomen area and skin of hindlimbs

  2. Open the abdominal cavity with blunt-end sterile scissors and remove the surface muscles and find the pelvic-hip joint (Figure 2).


    Figure 2. Find the pelvic-hip joint. Bone anatomy reference: http://www.informatics.jax.org/cookbook/figures/figure41.shtml

  3. Cut off the hind leg above the pelvic-hip joint with sharp sterile scissors (Figure 3). Cut off the tibia from the hind leg below the knee joint with sharp sterile scissors (Figure 4).


    Figure 3. Cut off the hind leg above the pelvic-hip joint


    Figure 4. Cut off the tibia at knee joint

  4. (Optional) If higher yield of bone marrow cells is needed, tibia can also be used for bone marrow cell isolation. Cut at the tibia ankle joint to dissect the tibia. The following procedures can be applied to both femur and tibia.
  5. Remove the muscles and residue tissues surrounding the femur with sterile forceps and scissors (Figure 5).


    Figure 5. Remove the muscles and residue tissues surrounding the femur

  6. Cut the femurs at both ends with sharp sterile scissors (Figure 6). Use a 23-gauge (some literature suggests 25-or 26-gauge) needle and a 10 cc syringe filled with ice-cold HBSS to flush the bone marrow out onto a 70 µm nylon cell strainer placed in a 50 ml Falcon conical tube (Figure 7). Use all the 10 ml HBSS or until the flow through turns white.


    Figure 6. Cut femurs at both ends


    Figure 7. Flush the bone marrow onto the cell strainer with HBSS

  7. (Optional) In case some residue bone marrow cells could not be flushed off (very few bone marrow visible in the flow through or the yield is significantly less, e.g., < 1 x 107 cells), scrape the inner surface of the femur with the needle and flush with extra ~5 ml HBSS (Figure 8).


    Figure 8. (Optional) Scrape the inner surface of femur with needle

  8. Smash the bone marrow through the cell strainer with a 5 ml plunger (Figure 9). Wash the strainer with another ~5 ml HBSS.


    Figure 9. Smash the bone marrow cells through the cell strainer with a 5 ml plunger

  9. Centrifuge cells at 1,500 rpm for 7 min at 4 °C. Discard the supernatant and blot on paper towel (Figure 10).


    Figure 10. Cell pellet before RBC buffer resuspension

  10. Resuspend the cell pellet with 1 ml RBC lysis buffer (for each mouse). Incubate for 5 min at room temperature or 2 min at 37 °C, and neutralize the lysis buffer by adding 5 ml FBS.
  11. Centrifuge cells at 1,500 rpm for 7 min at 4 °C. Discard the supernatant and blot on paper towel. Resuspend the cell pellet with appropriate media for the next step of assay such as 5 ml DMEM medium containing 10% FBS. Cells are then placed on ice.
  12. Count the bone marrow cells with a hemocytometer or a Beckman Z2 coulter counter. Cells are ready for assays or culture. Cells can stay viable on ice for at least 5 h. It is recommended to perform the experiment (culture or assays) right after isolation for best results.

Recipes

  1. RBC lysis buffer
    0.16 M NH4Cl, 10 mM KHCO3, and 0.13 mM EDTA, dissolved in sterile H2O and stored at 4 °C
    For 500 ml, 4.28 g NH4Cl, 0.5 g KHCO3, 0.024 g Disodium EDTA
    It is recommended to prepare fresh RBC lysis buffer for the experiment. RBC lysis buffer will be stable at 4 °C for at least 1 month.
  2. DMEM medium
    DMEM medium, high glucose, pyruvate, L-glutamine supplemented with 10% FBS
    Stored at 4 °C

Acknowledgments

This protocol was revised based on previous studies including the referenced articles below and was supported by an NIH grant (R21 MH099482) to Ning Quan.

References

  1. Madaan, A., Verma, R., Singh, A. T., Jain, S. K. and Jaggi, M. (2014). A stepwise procedure for isolation of murine bone marrow and generation of dendritic cells. J Biol Methods 1(1): e1.
  2. Weischenfeldt, J. and Porse, B. (2008). Bone marrow-derived macrophages (BMM): Isolation and applications. CSH Protoc 2008: pdb prot5080.

简介

骨髓是造血的部位,包含红细胞,粒细胞,单核细胞,树突状细胞,淋巴细胞和造血干细胞等混合血细胞。 以下方案提供了一种简单而快速的方法,用于在5ml培养基(1.6×10 4个细胞/μl)中分离来自小鼠股骨的骨髓免疫细胞(无红细胞),产量约为8×10 7个细胞。 研究特异性免疫细胞类型可能需要进一步的分离或流式细胞分析。

关键字:血, 流cytometry, 造血干细胞

材料和试剂

  1. 无菌纸巾
  2. 无菌手术垫(Direct Resource,目录号:19015742)
  3. 23号(或25-/26号)针(BD Biosciences,目录号:305145)
  4. 10ml注射器(BD Biosciences,目录号:309604)
  5. 70μm尼龙细胞过滤器(Falcon,目录号:352350) 注意:目前,"Corning,Falcon ? ,目录号:11995-065"。 />
  6. 50ml锥形管(Falcon,目录号:21008-940)
    />
  7. 5ml注射器柱塞(BD Biosciences,目录号:309646)
  8. 成年小鼠(> 6周,任何菌株)(例如C57BL/6)
  9. Hank's平衡盐溶液(HBSS),无钙,无镁,无酚红(Life Technologies,目录号:14175095)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:14175095" />
  10. DMEM培养基,高葡萄糖,丙酮酸盐,L-谷氨酰胺(Life Technologies,目录号:11995-065)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:11995-065"
  11. 70%乙醇
  12. 胎牛血清热灭活(FBS)(Sigma-Aldrich,目录号:F9665)
  13. 氯化铵(NH 4 Cl)(Sigma-Aldrich,目录号:213330)
  14. 碳酸氢钾(KHCO 3)(Sigma-Aldrich,目录号:237205)
  15. 乙二胺四乙酸二钠(Sigma-Aldrich,目录号:D2900000)
  16. RBC裂解缓冲液(参见配方)
  17. DMEM介质(参见配方)

设备

  1. 平头无菌剪刀(Thermo Fisher Scientific,Fisher Scientific,目录号:08-950)
  2. Sharp无菌剪刀(Thermo Fisher Scientific,Fisher Scientific,目录号:08-940)
  3. 无菌钳(Thermo Fisher Scientific,Fisher Scientific,目录号:08-890)
  4. (Thermo Fisher Scientific,Fisher Scientific,目录号:02-671-55A)或coulter Z2细胞和颗粒计数器(Beckman Coulter,目录号号码:383550)
  5. 冷冻离心机
  6. 无菌培养罩
  7. CO 啮齿动物安乐死室

程序

  1. 用CO 2安乐死小鼠,并在无菌罩中将小鼠放在无菌手术垫上。用70%乙醇灭菌小鼠腹部区域和后肢的皮肤(图1)。


    图1.小鼠腹部区域和后肢皮肤的消毒

  2. 用钝端无菌剪刀打开腹腔,取出表面肌肉,找到骨盆 - 髋关节(图2)。


    图2.找到骨盆 - 髋关节。 骨骼解剖参考: http://www.informatics.jax.org /cookbook/figures/figure41.shtml

  3. 用锋利的无菌剪刀剪断髋 - 髋关节上方的后腿(图3)。用锋利的无菌剪刀从膝关节下方的后腿切下胫骨(图4)

    图3.切断骨盆 - 髋关节上方的后腿


    图4.在膝关节处切断胫骨

  4. (可选)如果需要更高的骨髓细胞产量,胫骨也可用于骨髓细胞分离。切开胫骨踝关节解剖胫骨。以下程序可应用于股骨和胫骨。
  5. 用无菌镊子和剪刀去除股骨周围的肌肉和残留组织(图5)

    图5.去除股骨周围的肌肉和残留组织

  6. 用锋利的无菌剪刀剪两端的股骨(图6)。使用23号(一些文献建议25或26号)针和充满冰冷HBSS的10毫升注射器冲洗骨髓出70毫米尼龙细胞过滤器放置在一个50ml Falcon锥形管(图7)。使用所有10 ml HBSS或直到流量变为白色

    图6.两端切割股骨


    图7.用HBSS
    将骨髓冲洗到细胞滤网上
  7. (任选的)在一些残余物骨髓细胞不能被冲洗掉的情况下(在流过物中可见的骨髓非常少,或者产率明显更低,例如<1/10> <1×10 7 细胞),用针刮伤股骨的内表面并用额外?5ml的HBSS冲洗(图8)。


    图8.(可选)用针头刮伤股骨内表面

  8. 用5ml柱塞将骨髓粉碎通过细胞过滤器(图9)。用另一个?5ml的HBSS洗涤过滤器

    图9.用5 ml柱塞捣碎细胞过滤器的骨髓细胞

  9. 在4℃下以1,500rpm离心细胞7分钟。弃去上清液并在纸巾上吸干(图10)。


    图10. RBC缓冲液重悬之前的细胞沉淀

  10. 用1ml RBC裂解缓冲液(每只小鼠)重悬细胞沉淀。在室温孵育5分钟或在37℃孵育2分钟,并通过加入5ml FBS中和裂解缓冲液。
  11. 在4℃下以1,500rpm离心细胞7分钟。弃去上清液并在纸巾上吸干。用适当的培养基重悬细胞沉淀,用于下一步测定,例如5ml含有10%FBS的DMEM培养基。然后将细胞置于冰上。
  12. 用血细胞计数器或Beckman Z2 coulter计数器计数骨髓细胞。细胞准备用于测定或培养。细胞可以在冰上保持活力至少5小时。建议在分离后立即进行实验(培养或测定)以获得最佳结果。

食谱

  1. RBC裂解缓冲液
    0.16 M NH 4 Cl,10mM KHCO 3和0.13mM EDTA,溶解在无菌H 2 O中,并在4℃下储存。
    对于500ml,4.28g NH 4 Cl,0.5g KHCO 3,0.024g EDTA二钠
    建议为实验准备新鲜的RBC裂解缓冲液。 RBC裂解缓冲液在4℃下稳定至少1个月
  2. DMEM培养基
    DMEM培养基,高葡萄糖,丙酮酸盐,L-谷氨酰胺,补充有10%FBS 储存在4°C

致谢

该协议根据以前的研究,包括下面参考文章修订,并得到宁泉的NIH资助(R21 MH099482)的支持。

参考文献

  1. Madaan,A.,Verma,R.,Singh,A.T.,Jain,S.K.and Jaggi,M。(2014)。 分离小鼠骨髓和产生树突状细胞的分步程序 J Biol Methods 1(1):e1。
  2. Weischenfeldt,J。和Porse,B。(2008)。 骨髓来源的巨噬细胞(BMM):隔离和应用 CSH Protoc 2008:pdb prot5080。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Liu, X. and Quan, N. (2015). Immune Cell Isolation from Mouse Femur Bone Marrow. Bio-protocol 5(20): e1631. DOI: 10.21769/BioProtoc.1631.
  2. Liu, X., Yamashita, T., Chen, Q., Belevych, N., McKim, D. B., Tarr, A. J., Coppola, V., Nath, N., Nemeth, D. P., Syed, Z. W., Sheridan, J. F., Godbout, J. P., Zuo, J. and Quan, N. (2015). Interleukin 1 type 1 receptor restore: a genetic mouse model for studying interleukin 1 receptor-mediated effects in specific cell types. J Neurosci 35(7): 2860-2870.
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