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Isolation and Culture of Bone Marrow-derived Mast Cells
骨髓源肥大细胞的分离和培养   

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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). BMMCs are generally used for in vitro studies because of the high yield of mast cells generated and also because they can be generated from knockout and transgenic mice making this a good source to examine specific factors important for mast cell function. Due to the large yield of cells generated they are the cells of choice for reconstitution studies in mast cell knockout mice (Sur et al., 2007). Furthermore, they are more responsive to both allergic and non-allergic stimuli when compared to mast cell lines. The major disadvantage of BMMCs is that they are not fully matured when compared to mast cells generated or obtained from other sources. For example, compared to PCMCs [see the protocol “Isolation and Culture of Peritoneal Cell-derived Mast Cells” (Vukman et al., 2014)], BMMCs express a restricted range of TLRs and cytokines when stimulated with TLR ligands (Mrabet-Dahbi et al., 2009). 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 mouse bone marrow.

Keywords: Mast cells (肥大细胞), Cell culture (细胞培养), Primary cells (原代细胞), Bone marrow (骨髓), WEHI-3 (WEHI-3)

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. WEHI-3 conditioned medium generated from WEHI-3 cell line (ATCC, catalog number: TIB68 )
  4. Sterile phosphate buffered saline (PBS) (Life Technologies, Gibco®, catalog number: 14190 )
  5. IL-3 (BioLegend, catalog number: 432101 )
  6. IMDM with L-glutamine (Life Technologies, Gibco®, catalog number: 12440 )
  7. Fetal calf serum (FCS) (Life Technologies, Gibco®, catalog number: 10270 )
  8. Penicillin/streptomycin (Life Technologies, Gibco®, catalog number: 15140 )
  9. Mercapto-ethanol (Sigma-Aldrich, catalog number: M3148 )
  10. Trypan blue stain (Sigma-Aldrich, catalog number: T8154 )
  11. Complete IMDM (see Recipes)
  12. Growth factors (see Recipes)
  13. Kimura dye (see Recipes)

Equipment

  1. Sterile forceps
  2. Sterile scissors
  3. Sterile pipette
  4. Syringe (10 ml)
  5. Needle (19-, 21- and 27-gauge)
  6. Falcon tube (50 ml)
  7. Cell scraper (SARSTEDT AG, catalog number: 83.183 0)
  8. Filtropur S 0.45 filter (SARSTEDT AG, catalog number: 83.1826 )
  9. Petri dishes (100 x 20 mm)
  10. PD100 petri dish
  11. Water bath
  12. Centrifuge
  13. T75 Cell culture flask (SARSTEDT AG, catalog number: 83.1813.502 )
  14. T175 Cell culture flask (SARSTEDT AG, catalog number: 83.1812.502 )
  15. Haemocytometer
  16. Safety cabinet

Procedure

Note: All procedures are performed in a sterile environment in a class II safety cabinet.

  1. Preparing WEHI-3 conditioned medium
    1. Thaw cells from stock (which is stored in liquid nitrogen) by incubating cells in a water bath at 37 °C for 2 min.
    2. Resuspend cells in 37 °C complete IMDM as quick as possible. Final cell number has to be 2 x 105 cells/ml and culture cells in T75 tissue culture flask at 37 °C, 5% CO2.
    3. Passages: Transfer medium from tissue culture flask into a 50 ml Falcon tube.
    4. Scrape cells using sterile cell scraper.
    5. Transfer scraped cells into the same 50 ml Falcon tube.
    6. Count cells, the concentration has to be > 6 x 105 cell/ml.
    7. Centrifuge cells at 300 x g for 5 min.
    8. Transfer supernatant into new Falcon tube.
    9. Resuspend cells in fresh complete IMDM (2 x 105 cell/ml).
    10. Plate cells into new flasks (T75/T175).
    11. Centrifuge the supernatant again at 600 x g for 15 min.
    12. Transfer and filter supernatant into new Falcon tube.
    13. Take 0.5 ml for measurement of IL-3 concentrations by commercial ELISA. The concentration should range between 10 to 100 ng/ml and a minimum of 10 ng/ml should be used.
    14. Freeze supernatants at -20 °C.
    15. WEHI-3 conditioned medium can be harvested every 3-4 days following the steps A3-14. The cell number should not be over 2 x 106.

  2. Isolation and culture of BMMCs
    Day 0 [Passage (P) 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. Two sets of sterile tweezers and scissors are used, one for the outer skin of the mouse and the other for the removal of legs (tibia and femur).
    4. Make a small incision below the sternum of the mouse and cut the skin away from the front around the back to make a complete circle. Peel the fur away completely from the lower half of the mouse leaving the legs exposed.
    5. Use the second pair of scissors and tweezers. Holding the tail with the index and middle finger and the leg of interest between the ring finger and thumb, cut the muscle away from the hind leg around the hip in a straight cutting manner so as to make the hip-joint amenable for cutting.
    6. Cut the hip-joint to free the leg from the body of the mouse. Be careful not to cut and hence expose the bone marrow of the femur. Repeat this with the second leg of the mouse.
    7. Cut the ankle of each leg to remove the feet, and discard them.
    8. Place legs in 50 ml tubes on ice partially filled with IMDM until next step.
    9. Place legs onto one half of a PD100 petri dish and remove residual muscle, fat and connective tissue using forceps and scissors.
    10. Cut the knee joint to separate the femur and tibia.
    11. Use fresh pair of forceps and scissors to cut each bone in turn at either end to expose the bone marrow.
    12. Flush the bone marrow onto the dish using a 10 ml syringe filled with ice cold media with a 27-gauge needle. Repeat this for all bones.
    13. Use a 19-gauge needle to break up and suspend the bone marrow by pipetting.
    14. Transfer the bone marrow suspension in to a 50 ml tube and centrifuged at 300 x g for 10 min.
    15. Discard the supernatant and resuspend cells in culture medium (IMDM with 30% WEHI-3 conditioned medium; 20 ml medium/mouse) and transfer suspension into tissue-treated cell culture flask (T75).
    16. Culture cells at 37 °C, 5% CO2.
    Day 5 (P2)
    1. Transfer cell suspension into a 50 ml tube (avoid taking adherent cells!). Pellet the cells by centrifugation (300 x g, 10 min, 4 °C), discard supernatant. Loosen pellet by flicking the tube and resuspend cells in 20 ml fresh culture medium and transfer cell suspension into a new tissue-treated cell culture flask (T75).
    Day 8 (P3)
    1. Repeat procedure as on day 5.
    Day 12 (P4)
    1. Transfer cell suspension into a new big tissue treated cell culture flask (T175), (avoid taking over adherent cells!) and add 20 ml fresh culture medium.
    Day 15 (P5), day 22 (P7), day 29 (P9)
    1. Same procedure as on day 5 but use a big (T175) culture flask.
    Day 19 (P6), day 26 (P8)
    1. Transfer 20 ml of cell suspension into a 50 ml tube. Pellet the cells by centrifugation (300 x g, 10 min, 4 °C). Discard supernatant. Loosen pellet by flicking the tube and resuspend cells in 20 ml fresh culture medium and transfer cell suspension back into the cell culture flask.
    2. When cells are 4 weeks of age (P9) count cells and determine purity of mast cells via Kimura stain or FACS-analysis. If the cells are > 95% pure, they can be used for experiments (Figure 1). If the purity is less, they have to be cultured another week.
      Note: Do not exceed a cell density of 2.5 x 106.


      Figure 1. The purity of bone marrow-derived cultured mast cells is over 95% after 4 weeks of cultivation. Bone marrow cells from C57BL/6 mouse were cultured for 4 weeks in IMDM (with 10% FCS, 100 U/ml penicillin/streptomycin and 50 μM 2-mercaptoethanol) in the presence of 30% WEHI-3 conditioned medium. Cell number (A) and purity (B) was determined by every passage, twice a week with trypan blue and Kimura staining. Data are presented as the mean ± SD of nine independent experiments.

Recipes

  1. Complete IMDM
    IMDM (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 IMDM prior to use)
    30% WEHI-3 conditioned medium
  3. Kimura dye
    Toluidine blue solution (50 ml) (0.5 mg/ml Toluidine blue, 18 g/l NaCl and 22% ethanol)
    Saturated saponin (2.27 ml) (4 mg/ml saponin in ethanol)
    NaH2PO4 solution (22.7 ml) (60 mM)

Acknowledgments

The protocol described here was adapted from Sur et al. (2007) and 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. 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.
  3. 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.
  4. 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.
  5. 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.
  6. Sur, R., Cavender, D. and Malaviya, R. (2007). Different approaches to study mast cell functions. Int Immunopharmacol 7(5): 555-567.
  7. 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.
  8. 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.
  9. 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.

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)。 BMMC通常用于体外研究,因为所产生的肥大细胞的高产率,并且还因为它们可以从敲除和转基因小鼠产生,使得它成为检查对肥大细胞功能重要的特定因子的良好来源。由于产生的细胞的大量产生,它们是在肥大细胞敲除小鼠中重建研究中选择的细胞(Sur等人,2007)。此外,当与肥大细胞系相比时,它们对过敏性和非过敏性刺激更敏感。 BMMC的主要缺点是当与从其它来源产生或获得的肥大细胞相比时,它们没有完全成熟。例如,与PCMC相比[参见协议"腹膜细胞源性肥大细胞的分离和培养" "(Vukman等人,2014)],当用TLR配体刺激时,BMMC表达限制性范围的TLR和细胞因子(Mrabet-Dahbi等人,2009)。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,我们描述了小鼠骨髓中鼠肥大细胞的分离和培养的协议。

关键字:肥大细胞, 细胞培养, 原代细胞, 骨髓, WEHI-3

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. WEHI-3条件培养基由WEHI-3细胞系(ATCC,目录号:TIB68)产生
  4. 无菌磷酸盐缓冲盐水(PBS)(Life Technologies,Gibco ,目录号:14190)
  5. IL-3(BioLegend,目录号:432101)
  6. IMDM与L-谷氨酰胺(Life Technologies,Gibco ,目录号:12440)
  7. 胎牛血清(FCS)(Life Technologies,Gibco ,目录号:10270)
  8. 青霉素/链霉素(Life Technologies,Gibco ,目录号:15140)
  9. 巯基乙醇(Sigma-Aldrich,目录号:M3148)
  10. 台盼蓝染料(Sigma-Aldrich,目录号:T8154)
  11. 完成IMDM(请参阅配方)
  12. 生长因子(参见配方)
  13. 木村染料(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(19,21和27号)
  6. Falcon管(50ml)
  7. 细胞刮刀(SARSTEDT AG,目录号:83.1830)
  8. Filtropur S 0.45过滤器(SARSTEDT AG,目录号:83.1826)
  9. 培养皿(100x20mm)
  10. PD100培养皿
  11. 水浴
  12. 离心机
  13. T75细胞培养瓶(SARSTEDT AG,目录号:83.1813.502)
  14. T175细胞培养瓶(SARSTEDT AG,目录号:83.1812.502)
  15. 血细胞计数器
  16. 安全柜

程序

注意:所有程序均在无菌环境中在II级安全柜中进行。

  1. 制备WEHI-3条件培养基
    1. 通过在37℃的水浴中孵育细胞2分钟,使来自储备液(其储存在液氮中)的细胞解冻。
    2. 重悬细胞在37℃完全IMDM尽快。 最终细胞数必须是2×10 5个细胞/ml和在37℃,5%CO 2下的T75组织培养瓶中的培养细胞。
    3. 通道:将培养基从组织培养瓶转移到50ml Falcon管中
    4. 用无菌细胞刮刀刮细胞
    5. 将刮取的细胞转移到同一个50ml Falcon管中
    6. 计数细胞,浓度必须> 6×10 5细胞/ml
    7. 以300×g离心细胞5分钟。
    8. 将上清转移到新的Falcon管中
    9. 将细胞重悬在新鲜的完全IMDM(2×10 5细胞/ml)中
    10. 将板细胞转移到新烧瓶(T75/T175)中
    11. 将上清液再次以600×g离心15分钟
    12. 将上清液转移并过滤到新的Falcon管中
    13. 取0.5 ml用于通过商业ELISA测量IL-3浓度。 浓度应介于10至100 ng/ml之间,最低浓度应为10 ng/ml
    14. 在-20℃下冷冻上清液
    15. WEHI-3条件培养基可以在步骤A3-14之后每3-4天收获。 单元格编号不应超过2 x 10 6

  2. BMMC的分离和培养
    第0天[通道(P)1]
    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. 使用两套无菌镊子和剪刀,一个用于鼠标的外皮,另一个用于去除腿(胫骨和股骨)。
    4. 在小鼠的胸骨下面做一个小切口,切开皮肤远离前面的后面,使一个完整的圆。 将毛皮完全从鼠标的下半部分剥离,使腿露出来。
    5. 使用第二对剪刀和镊子。 用食指和中指以及无名指和拇指之间的感兴趣的腿握住尾部,以直线切割方式将肌肉远离围绕髋部的后腿切开,以使髋关节适于切割。
    6. 切开髋关节,以从鼠标的身体释放腿。 小心不要切开,因此暴露股骨的骨髓。 对鼠标的第二个腿重复此操作。
    7. 剪下每条腿的脚踝以移除脚,并丢弃它们。
    8. 将腿放置在冰上的部分填充有IMDM的50ml管中,直到下一步骤
    9. 将腿放在PD100培养皿的一半上,并使用镊子和剪刀去除残余的肌肉,脂肪和结缔组织。
    10. 切开膝关节,分开股骨和胫骨
    11. 使用新鲜的钳子和剪刀在每端依次切割骨头,暴露骨髓
    12. 使用填充有具有27号针的冰冷的介质的10ml注射器将骨髓冲洗到培养皿上。 对所有骨骼重复此操作。
    13. 使用19号针头打破并通过移液器悬挂骨髓。
    14. 将骨髓悬浮液转移到50ml管中,并在300×g离心10分钟。
    15. 弃去上清液并将细胞重悬在培养基(含有30%WEHI-3条件培养基的IMDM; 20ml培养基/小鼠)中,并将悬浮液转移到组织处理的细胞培养瓶(T75)中。
    16. 在37℃,5%CO 2培养细胞
    第5天(P2)
    1. 将细胞悬液转移到50ml管(避免服用贴壁细胞!)。 通过离心(300×g,10分钟,4℃)沉淀细胞,弃去上清液。 通过轻轻管松开细胞和重悬细胞在20ml新鲜培养基中,并将细胞悬浮液转移到新的组织处理的细胞培养瓶(T75)。
    第8天(P3)
    1. 重复与第5天相同的步骤。
    第12天(P4)
    1. 将细胞悬液转移到新的大组织处理的细胞培养瓶(T175),(避免接管贴壁细胞!),并加入20ml新鲜培养基。
    第15天(P5),第22天(P7),第29天(P9)
    1. 与第5天相同的程序,但使用大(T175)培养瓶。
    第19天(P6),第26天(P8)
    1. 转移20ml细胞悬浮液到50ml管中。通过离心(300×g,10分钟,4℃)沉淀细胞。弃去上清液。通过轻弹管松开细胞团,将细胞重悬于20ml新鲜培养基中,并将细胞悬浮液转移回细胞培养瓶。
    2. 当细胞为4周龄(P9)计数细胞并通过Kimura染色或FACS分析确定肥大细胞的纯度。如果细胞> 95%纯,它们可用于实验(图1)。如果纯度较低,则必须再培养一周。
      注意: 不要超过2.5 x 10 6 的单元格密度。


      图1.培养4周后,骨髓来源的培养的肥大细胞的纯度超过95%。将来自C57BL/6小鼠的骨髓细胞在IMDM(含10%FCS)中培养4周, ,100U/ml青霉素/链霉素和50μM2-巯基乙醇)中,在30%WEHI-3条件培养基的存在下。 每次传代测定细胞数(A)和纯度(B),每周两次,用台盼蓝染色和Kimura染色。 数据表示为9次独立实验的平均值±SD

食谱

  1. 完成IMDM
    IMDM(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(在使用前加入IMDM)
    30%WEHI-3条件培养基
  3. 木村染料
    甲苯胺蓝溶液(50ml)(0.5mg/ml甲苯胺蓝,18g/l NaCl和22%乙醇)
    饱和皂苷(2.27ml)(4mg/ml皂甙在乙醇中)
    NaH 2 PO 4溶液(22.7ml)(60mM)中。

致谢

这里描述的方案改编自Sur等人(2007)和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. 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。
  3. 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。
  4. 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。
  5. 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。 Immunol 167(4):2250-2256。
  6. 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
  7. 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免疫应答的能力。 J Immunol 190(6):2873-2879。
  8. Vukman,K.V.,Metz,M.,Maurer,M。和O'Neill,S.M。(2014)。 腹膜细胞衍生的肥大细胞的分离和培养 生物协议 4(4):e1052。
  9. 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。
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引用: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. DOI: 10.21769/BioProtoc.1053.
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Barbara Summers
Weill COrnell Medical College
Can you do a western on BMMC using anti FCεR1α to prove that your cells are mast cells or is FACS only possible with this antibody.
3/31/2015 5:57:49 AM Reply
Krisztina Vukman
Dublin City University

You can do a western, but I think with that method you can only prove that you have mast cells in your cell culture, but are not able to check purity. I think Kimura stain is the easiest way to check purity. I ususally use facs, because it is very specific and reliable.

4/6/2015 11:25:12 PM


Barbara Summers
Weill COrnell Medical College
Why did you use Kimura staining? Is it specific for only mast cells?
3/25/2015 12:15:29 PM Reply
Krisztina Vukman
Dublin City University

Kimura stain contains toluidine blue and it is often used to identify mast cells, because they stain heparin in their cytoplasmic granules. I tried it with some other bone marrow-derived cells and it was specific for mast cells. (Krisztina)

3/30/2015 1:00:35 AM


Barbara Summers
Weill COrnell Medical College
Our lab tried your protocol 3 times. Each time, 90% of the cells adhered to the flask. We had to hit the flask very hard to get cells in suspension. When replated, the cells stuck again. Any idea why we saw mostly adherent cells? After the first 5 days, will most of the cells be adherent?
11/21/2014 5:34:27 AM Reply
Sandra O’Neill
Biotechnology Department, Dublin City University, Ireland

When feeding the cells it is important to take only the non-adherent cells. The cells adhered to the flask are not mast cells, so try not to get them in suspension. The mast cells yield in the begining is very low, but it will be 30-40 million by passage 9, and there will be less and less adherent cells. We also use flasks for non-adherent cells (cat.no. in the protocol).

11/25/2014 12:18:28 AM


Barbara Summers
Weill COrnell Medical College
Does it matter how old the mouse is? Will it work on a 5 month old mouse? How will the yield compare to a 4 week old mouse?
8/6/2014 8:43:34 AM Reply
Sandra O’Neill
Biotechnology Department, Dublin City University, Ireland

We did not see any difference within BMMCs from old and young mice. In other labs they prefer to use old (few month old) mice.

11/25/2014 12:06:03 AM