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Isolation and Culture of Peritoneal Cell-derived Mast Cells
来源于腹水的肥大细胞分离和培养   

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参见作者原研究论文

本实验方案简略版
The Journal of Immunology
Mar 2013

Abstract

The generation of mast cells for in vitro studies comes from a variety of sources including mast cell lines (MC/9) (McCurdy et al., 2001), bone marrow-derived mast cells (BMMCs) (Supajatura et al., 2001), skin-derived mast cells (FSMCs) (Matsushima et al., 2004), peritoneal-derived mast cells (PMCs) (Hochdorfer et al., 2011) and peritoneal cell-derived cultured mast cells (PCMCs) (Vukman et al., 2012). PCMCs are generally used for in vitro studies because they are a more mature source of mast cells when compared to mast cells generated or obtained from other sources. They can differ, for example, in their pro-inflammatory responses to bacterial antigens and toll like receptors (TLRs) ligands (Mrabet-Dahbi et al., 2009). In comparison to BMMCs [see the protocol “Isolation and Culture of Bone Marrow-derived Mast Cells” (Vukman et al., 2014)] or mast cell lines they express a wider range of TLRs, and secrete significantly more cytokines when stimulated with TLR ligands (Mrabet-Dahbi et al., 2009). Therefore, when examining pro-inflammatory responses, mast cells generated from cells obtained from the peritoneal cavity give stronger responses. PCMCs can also be generated from knockout and transgenic mice making them a good source to examine specific factors important for mast cell function. However, due to the low yield of cells generated using this method (1 million per mouse) their use is restricted and therefore in most studies more than one source of mast cells may be required. The different sources of mast cells can display phenotypical and functional differences and therefore it is important that when designing an experiment, the correct cellular source is obtained. Here, we describe a protocol for the isolation and culture of murine mast cells from peritoneal cavity cells.

Keywords: Mast cells (肥大细胞), Peritoneal cavity (腹膜腔), Peritoneal lavage (腹腔灌洗), Cell culture (细胞培养)

Materials and Reagents

  1. C57BL/6 mice or mouse model of choice (Harlan Laboratories, catalog number: 057 ; Charles River Laboratories International, catalog number: BLCSIFE49D )
  2. Industrial methylated spirit (IMS) (Lennox Laboratory Supplies, catalog number: CRTS10330716 )
  3. Sterile phosphate buffered saline (PBS) (Life Technologies, Gibco®, catalog number: 14190 )
  4. RPMI 1640 medium (Life Technologies, Gibco®, catalog number: 31870 )
  5. Fetal calf serum (FCS) (Life Technologies, Gibco®, catalog number: 10270 )
  6. Penicillin/streptomycin (Life Technologies, Gibco®, catalog number: 15140 )
  7. 2-Mercapto-ethanol (Sigma-Aldrich, catalog number: M3148 )
  8. L-glutamine (Sigma-Aldrich, catalog number: G7513 )
  9. Recombinant stem cell factor (SCF) (Sigma-Aldrich, catalog number: S9915 )
  10. Recombinant mouse interleukin-3 (IL-3) (Merck KGaA, catalog number: 407631 )
  11. Trypan blue stain (Sigma-Aldrich, catalog number: T8154 )
  12. APC-conjugated c-kit (eBioscience, catalog number: 17-1171 )
  13. Fitc congugted-FcεRI (eBioscience, catalog number: 11-5898 )
  14. Complete RPMI (see Recipes)
  15. Growth factors (see Recipes)
  16. Kimura dye (see Recipes)
  17. Toluidine blue solution (see Recipes)
  18. Saturated saponin (see Recipes)
  19. NaH2PO4 solution (see Recipes)

Equipment

  1. Sterile forceps
  2. Sterile scissors
  3. Sterile pipette
  4. Syringe (10 ml)
  5. Needle (19-gauge)
  6. Falcon tube (15 or 50 ml)
  7. Water bath
  8. pH meter
  9. Centrifuge
  10. 37 °C, 5% CO2 incubator
  11. T25 Cell culture flask (SARSTEDT AG, catalog number: 83.1810.502 )
  12. Haemocytometer
  13. Safety cabinet

Procedure

Note: All procedures are done in sterile environment in a safety cabinet.

Day 1

  1. Kill mouse by cervical dislocation.
  2. Spray mouse thoroughly with 70% alcohol (or IMS) and lay down on 70% alcohol soaked paper.
  3. Make a small incision below the sternum of the mouse and peel the fur back from the chest. Be careful not to puncture the peritoneum. Use sterile scissors and tweezers.
  4. Inject 1 ml air and 10 ml ice cold sterile PBS into the peritoneal cavity of the mouse using 10 ml syringe with a 19-gauge needle.
    Note: 1:1 mixture of air and PBS can also be used to avoid leaking.
  5. Do not remove the needle.
  6. Give 30 sec massage on two sides of the mouse with fingers. Be careful not to push too hard the mouse.
  7. Collect the PBS with the cells into a Falcon tube with the syringe.
    Note: Needle can be replaced for a bigger one to collect cells from the peritoneal cavity.
  8. Open the peritoneal cavity with new scissor and forceps and collect the rest of the PBS with a sterile pipette.
  9. Place the Falcon tube onto ice until centrifugation.
  10. Centrifuge cells at 4 °C with 300 x g for 10 min.
  11. Resuspend cells in 5 ml PBS per mouse.
  12. Centrifuge cells at 4 °C with 300 x g for 10 min.
  13. Resuspend cells in 5 ml medium [with 10 ng/ml IL-3 and 30 ng/ml SCF per two mice (approximately 1 million cells obtained per mouse)] and transfer them into a small culture flask.
  14. Put the flasks into the incubator (37 °C, 5% CO2).

Day 3

  1. Remove non-adherent cells by discarding the medium and add 5 ml fresh medium (with 10 ng/ml IL-3 and 30 ng/ml SCF) to every flask.
  2. Put them back into the incubator (37 °C, 5% CO2).

Day 6

  1. Add 5 ml fresh medium (with 10 ng/ml IL-3 and 30 ng/ml SCF) to every flask.

Day 9 or 10

  1. PCMCs are the non-adherent cells and can be used for experiment.
  2. Cell number and viability can be measured with trypan blue staining in haemocytometer.
  3. Purity can be tested with Kimura staining by mixing the cell suspension and Kimura dye (1:1) and after 5 min at 37 °C count cells under a light microscope. Mast cells will be stained red/purple (Kimura et al., 1973).
    Note: Expected cell number is 0.5-1.5 x 106 PCMC/mouse after culturing. Purity is > 95% (Figure 1).


    Figure 1. The purity of peritoneal-derived cultured mast cells is over 95% after 9 days of cultivation. Peritoneal cells were cultured for 9 days in complete RPMI medium in the presence of 10 ng/ml IL-3 and 30 ng/ml SCF. Purity was assessed on the basis of APC-conjugated c-kit and Fitc congugted-FcεRI cell surface expression measured by flow cytometry as previously described (Vukman et al., 2013). A: unstained control; B: double stained sample.

Recipes

  1. Complete RPMI
    RPMI 1640 (500 ml)
    10% FCS (50 ml per 450 ml of IMDM)
    5 ml Penicillin (100 U/ml)/Streptomycin (100 μg/ml)
    1 M 2-mercaptoethanol (add 25 μl to 500 ml for 50 μM final concentration)
  2. Growth factors (add to complete media prior to use)
    Mouse SCF (30 μg/ml)
    Mouse IL-3 (10 μg/ml)
  3. Kimura dye
    Toluidine blue solution (50 ml)
    Saturated saponin (2.27 ml)
    NaH2PO4 solution (22.7 ml)
  4. Toluidine blue solution
    Toluidine blue (25 mg) (0.5 mg/ml)
    1.8% NaCl (18 g/L) (25 ml) (9 g/L)
    Ethanol (100%) (11 ml) (22%)
    Total 50 ml
  5. Saturated saponin
    Saponin (200 mg) (4 mg/ml)
    Ethanol (50%) (50 ml)
    Total 50 ml
    Note: Heat and vortex for 30 min.
  6. NaH2PO4 solution
    60 mM NaH2PO4 (1.03 g)
    Total 100 ml (pH 6.4)

Acknowledgments

The protocol described here was used in and adapted from the following publication (Vukman et al., 2012).

References

  1. Hochdorfer, T., Kuhny, M., Zorn, C. N., Hendriks, R. W., Vanhaesebroeck, B., Bohnacker, T., Krystal, G. and Huber, M. (2011). Activation of the PI3K pathway increases TLR-induced TNF-alpha and IL-6 but reduces IL-1beta production in mast cells. Cell Signal 23(5): 866-875.
  2. Kimura I., Moritani Y. and Tanizaki Y. (1973). Basophils in bronchial asthma with reference to regain-type allergy. Clin Allergy 3(2):195-202.
  3. Matsushima, H., Yamada, N., Matsue, H. and Shimada, S. (2004). TLR3-, TLR7-, and TLR9-mediated production of proinflammatory cytokines and chemokines from murine connective tissue type skin-derived mast cells but not from bone marrow-derived mast cells. J Immunol 173(1): 531-541.
  4. McCurdy, J. D., Lin, T. J. and Marshall, J. S. (2001). Toll-like receptor 4-mediated activation of murine mast cells. J Leukoc Biol 70(6): 977-984.
  5. Mrabet-Dahbi, S., Metz, M., Dudeck, A., Zuberbier, T. and Maurer, M. (2009). Murine mast cells secrete a unique profile of cytokines and prostaglandins in response to distinct TLR2 ligands. Exp Dermatol 18(5): 437-444.
  6. Supajatura, V., Ushio, H., Nakao, A., Okumura, K., Ra, C. and Ogawa, H. (2001). Protective roles of mast cells against enterobacterial infection are mediated by Toll-like receptor 4. J Immunol 167(4): 2250-2256.
  7. Sur, R., Cavender, D. and Malaviya, R. (2007). Different approaches to study mast cell functions. Int Immunopharmacol 7(5): 555-567.
  8. Vukman, K. V., Visnovitz, T., Adams, P. N., Metz, M., Maurer, M. and O'Neill, S. M. (2012). Mast cells cultured from IL-3-treated mice show impaired responses to bacterial antigen stimulation. Inflamm Res 61(1): 79-85.
  9. Vukman, K. V., Adams, P. N., Metz, M., Maurer, M. and O'Neill, S. M. (2013). Fasciola hepatica tegumental coat impairs mast cells' ability to drive Th1 immune responses. J Immunol 190(6): 2873-2879.
  10. Vukman, K. V., Metz, M., Maurer, M. and O'Neill, S. M. (2014). Isolation and culture of bone marrow-derived mast cells. Bio-protocol 4(4): e1053.

简介

用于体外研究的肥大细胞的产生来自多种来源,包括肥大细胞系(MC/9)(McCurdy等人,2001),骨髓 - 源自人的肥大细胞(BMMC)(Supajatura等人,2001),皮肤来源的肥大细胞(FSMC)(Matsushima等人,2004),腹膜来源的唾液细胞(PMC)(Hochdorfer等人,2011)和腹膜细胞衍生的培养的肥大细胞(PCMC)(Vukman等人,2012)。 PCMC通常用于体外研究,因为它们与从其它来源产生或获得的肥大细胞相比是更成熟的肥大细胞来源。它们可以不同,例如,它们对细菌抗原和toll样受体(TLR)配体的促炎反应(Mrabet-Dahbi等人,2009)。与BMMC相比[参见协议"骨髓衍生肥大细胞的分离和培养(Vukman等人,2014)]或肥大细胞系,它们表达更宽范围的TLR,并且当用TLR配体刺激时分泌显着更多的细胞因子(Mrabet-Dahbi& et al。,,2009)。因此,当检查促炎反应时,从获自腹膜腔的细胞产生的肥大细胞产生更强的反应。 PCMC也可以从敲除和转基因小鼠产生,使它们成为检查对肥大细胞功能重要的特定因子的良好来源。然而,由于使用该方法产生的细胞产量低(每只小鼠100万个),它们的使用受到限制,因此在大多数研究中可能需要超过一个肥大细胞来源。肥大细胞的不同来源可以显示表型和功能差异,因此重要的是,当设计实验时,获得正确的细胞来源。在这里,我们描述了从腹腔腔细胞分离和培养鼠肥大细胞的协议。

关键字:肥大细胞, 腹膜腔, 腹腔灌洗, 细胞培养

材料和试剂

  1. C57BL/6小鼠或选择的小鼠模型(Harlan Laboratories,目录号:057; Charles River Laboratories International,目录号:BLCSIFE49D)
  2. 工业甲基化酒精(IMS)(Lennox Laboratory Supplies,目录号:CRTS10330716)
  3. 无菌磷酸盐缓冲盐水(PBS)(Life Technologies,Gibco ,目录号:14190)
  4. RPMI 1640培养基(Life Technologies,Gibco ,目录号:31870)
  5. 胎牛血清(FCS)(Life Technologies,Gibco ,目录号:10270)
  6. 青霉素/链霉素(Life Technologies,Gibco ,目录号:15140)
  7. 2-巯基乙醇(Sigma-Aldrich,目录号:M3148)
  8. L-谷氨酰胺(Sigma-Aldrich,目录号:G7513)
  9. 重组干细胞因子(SCF)(Sigma-Aldrich,目录号:S9915)
  10. 重组小鼠白细胞介素3(IL-3)(Merck KGaA,目录号:407631)
  11. 台盼蓝染料(Sigma-Aldrich,目录号:T8154)
  12. APC偶联的c-kit(eBioscience,目录号:17-1171)
  13. Fitc congugted-FcεRI(eBioscience,目录号:11-5898)
  14. 完成RPMI(参见配方)
  15. 生长因子(参见配方)
  16. 木村染料(见配方)
  17. 甲苯胺蓝溶液(参见配方)
  18. 饱和皂苷(见配方)
  19. NaH 2 PO 4溶液(参见配方)。

设备

  1. 无菌钳
  2. 无菌剪刀
  3. 无菌移液器
  4. 注射器(10ml)
  5. 针(19号)
  6. Falcon管(15或50ml)
  7. 水浴
  8. pH计
  9. 离心机
  10. 37℃,5%CO 2培养箱
  11. T25细胞培养瓶(SARSTEDT AG,目录号:83.1810.502)
  12. 血细胞计数器
  13. 安全柜

程序

注意:所有程序均在无菌环境下在安全柜中进行。

第1天

  1. 通过颈椎脱臼杀死老鼠。
  2. 用70%酒精(或IMS)彻底地喷洒小鼠,放在70%酒精浸泡的纸上
  3. 在小鼠的胸骨下方做一个小切口,将毛皮从胸部剥离。 小心不要刺伤腹膜。 使用无菌剪刀和镊子
  4. 使用带有19号针的10ml注射器,将1ml空气和10ml冰冷无菌PBS注入小鼠的腹膜腔。
    注意:1:1的空气和PBS的混合物也可以用来避免泄漏。
  5. 不要取下针。
  6. 用手指给鼠标两侧30秒的按摩。 小心不要用力过猛。
  7. 用注射器将带有细胞的PBS收集到Falcon管中。
    注意:可以更换一个更大的针,从腹膜腔收集细胞。
  8. 用新的剪刀和镊子打开腹腔,用无菌移液管收集PBS的其余部分。
  9. 将Falcon管置于冰上直至离心
  10. 在4℃下用300×g离心细胞10分钟
  11. 重悬细胞在每只小鼠5ml PBS中
  12. 在4℃下用300×g离心细胞10分钟
  13. 将细胞重悬于5ml培养基[每2只小鼠10ng/ml IL-3和30ng/ml SCF(每只小鼠获得约100万细胞)]中,并将其转移到小培养瓶中。
  14. 将烧瓶放入培养箱(37℃,5%CO 2)中

第3天

  1. 通过弃去培养基除去非贴壁细胞,并向每个烧瓶中加入5ml新鲜培养基(含10ng/ml IL-3和30ng/ml SCF)。
  2. 将它们放回培养箱(37℃,5%CO 2)中。

第6天

  1. 向每个烧瓶中加入5ml新鲜培养基(含10ng/ml IL-3和30ng/ml SCF)

第9或10天

  1. PCMC是非粘附细胞,可用于实验
  2. 细胞数和存活率可以用血球计中的台盼蓝染色测量
  3. 可以通过将细胞悬浮液和Kimura染料(1:1)混合并在37℃下在光学显微镜下计数细胞5分钟后用Kimura染色测试纯度。肥大细胞将被染成红色/紫色(Kimura等人,1973)。
    注意:培养后预期的细胞数为0.5-1.5×10 6 PCMC /小鼠。纯度> 95%(图1)。


    图1.培养9天后,腹膜来源的培养的肥大细胞的纯度超过95%。在10ng/ml的IL-10存在下,在完全RPMI培养基中培养腹膜细胞9天, 3和30ng/ml SCF。如先前所述,通过流式细胞术测量APC-缀合的c-kit和Fitc congugted-FcεRI细胞表面表达的基础上评估纯度(Vukman等人,2013)。 A:未染色对照; B:双重染色样品

食谱

  1. 完成RPMI
    RPMI 1640(500ml) 10%FCS(50ml/450ml IMDM) 5ml青霉素(100U/ml)/链霉素(100μg/ml) 1 M 2-巯基乙醇(对于50μM终浓度,加入25μl至500ml)
  2. 生长因子(使用前添加到完全培养基中)
    小鼠SCF(30μg/ml)
    小鼠IL-3(10μg/ml)
  3. 木村染料
    甲苯胺蓝溶液(50ml) 饱和皂苷(2.27ml)
    NaH 2 PO 4溶液(22.7ml)中
  4. 甲苯胺蓝溶液
    甲苯胺蓝(25mg)(0.5mg/ml)
    1.8%NaCl(18g/L)(25ml)(9g/L) 乙醇(100%)(11ml)(22%) 总共50ml ml
  5. 饱和皂苷
    皂苷(200mg)(4mg/ml)
    乙醇(50%)(50ml) 总共50ml ml
    注意:加热和涡旋30分钟。
  6. NaH 2 PO 4溶液
    60mM NaH 2 PO 4(1.03g)
    总共100ml(pH 6.4)

致谢

本文描述的方案用于和改编自以下出版物(Vukman等人,2012)。

参考文献

  1. Hochdorfer,T.,Kuhny,M.,Zorn,C.N.,Hendriks,R.W.,Vanhaesebroeck,B.,Bohnacker,T.,Krystal,G.and Huber, PI3K通路的激活增加TLR诱导的TNF-α和IL-6,但减少IL-1beta 生产在肥大细胞中。 细胞信号 23(5):866-875。
  2. Kimura I.,Moritani Y.and Tanizaki Y.(1973)。 Basophils in bronchial asthma with reference to regain-type allergy。 Clin Allergy 3(2):195-202。
  3. Matsushima,H.,Yamada,N.,Matsue,H。和Shimada,S。(2004)。 TLR3-,   TLR7-和TLR9介导的促炎细胞因子的产生 来自鼠结缔组织型皮肤来源的肥大细胞的趋化因子 但不是来自骨髓来源的肥大细胞。 J Immunol 173(1):531-541。
  4. McCurdy,J.D.,Lin,T.J.and Marshall,J.S。(2001)。 Toll样受体4介导的鼠肥大细胞活化。 Leukoc Biol 70(6):977-984。
  5. Mrabet-Dahbi,S.,Metz,M.,Dudeck,A.,Zuberbier,T.and Maurer,M。(2009)。 鼠肥大细胞分泌一个独特的细胞因子和前列腺素响应不同的TLR2配体配置文件。 Exp Dermatol 18(5):437-444。
  6. Supajatura,V.,Ushio,H.,Nakao,A.,Okumura,K.,Ra,C.and Ogawa,H。(2001)。 肥大细胞对肠道细菌感染的保护作用由Toll样受体4介导。
  7. Sur,R.,Cavender,D.and Malaviya,R。(2007)。 研究肥大细胞功能的不同方法 Int Immunopharmacol 7(5):555-567
  8. Vukman,K.V.,Visnovitz,T.,Adams,P.N.,Metz,M.,Maurer,M。和O'Neill,S.M。(2012)。 从IL-3处理的小鼠培养的肥大细胞显示对细菌抗原刺激的受损反应。 Inflamm Res 61(1):79-85
  9. Vukman,K.V.,Adams,P.N.,Metz,M.,Maurer,M。和O'Neill,S.M。(2013)。 Fasciola hepatica tegumental coat损害肥大细胞驱动Th1免疫反应的能力 。 J Immunol 190(6):2873-2879。
  10. Vukman,K.V.,Metz,M.,Maurer,M。和O'Neill,S.M。(2014)。 骨髓衍生的肥大细胞的分离和培养 生物协议 4(4):e1053。
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引用:Vukman, K. V., Metz, M., Maurer, M. and O’Neill, S. M. (2014). Isolation and Culture of Peritoneal Cell-derived Mast Cells. Bio-protocol 4(4): e1052. DOI: 10.21769/BioProtoc.1052.
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René Bigirimana
Argenx
Number of cells obtained were too low.
10/1/2018 4:09:45 AM Reply
Krisztina Vukman
Dublin City University

Cell number is depend on many things:
- From old mice, you can get more cells
- concentration and source of IL-3 is important as well
- During peritoneal lavage the "massage" step is very important
- Cells are quite sensitive. They do not like harsh pipetting, changes in temperature or pH, long centrifugation, etc.

10/1/2018 4:54:59 AM


Barbara Summers
Weill COrnell Medical College
On step 15 it says to discard non-adherent cells. Did you mean to spin down media and resuspend non adherent cells in fresh media and then plate into a new flasks?

Thanks
Barbara
12/11/2014 5:52:50 AM Reply
Krisztina Vukman
Dublin City University

Yes

10/1/2018 4:49:14 AM