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 JoVE Biology

分离和富集大鼠间质干细胞(MSCS)及衍生的干细胞分离单细胞集落

1, 1

1Department of Chemical Engineering and Materials Science, City of Hope Cancer Center

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    Summary

    大鼠MSCs从股骨和胫骨分离,然后通过磁性细胞分选富集。排序细胞被证实为流式细胞仪检测表面标志物表达。这些细胞克隆密度培养,形成单菌落,然后这些菌落克隆气瓶分开。

    Date Published: 3/22/2010, Issue 37; doi: 10.3791/1852

    Cite this Article

    Zhang, L., Chan, C. Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs. J. Vis. Exp. (37), e1852, doi:10.3791/1852 (2010).

    Abstract

    干细胞是一个成人干细胞的人口,这是一个治疗应用前途源。这些细胞可以从骨髓中分离,可以很容易地从造血干细胞(HSCs)由于塑料坚持分开。此协议介绍了如何从大鼠股骨和胫骨的干细胞分离。分离出的细胞对两个干细胞表面标志物CD54和磁性细胞分选CD90的进一步丰富。表面标志物CD54和CD90的表达,然后通过流式细胞仪分析证实。 HSC的标记CD45的也包括在内,以检查是否排序干细胞,造血干细胞耗尽。干细胞是自然相当异构。有不同的形状,增殖和分化能力的细胞亚群。所有这些亚群表达已知的干细胞标记,并没有独特的标记尚未确定不同的亚群。因此,分离出不同的亚群的一种替代方法是使用克隆气瓶分离出单细胞集落来源的细胞。然后可以从单一的殖民地所得的细胞培养和单独评价。

    Protocol

    1。大鼠MSCs的分离

    从6-8周龄只Sprague - Dawley雌性大鼠如前所述1,2间质干细胞分离。隔离的MSCs能坚持到塑料表面,并轻松地扩展在体外培养。

    1. 动物被放入一个麻醉室,麻醉,大约5分钟。在麻醉,观察闪烁,呼吸和运动活动的速度。立即从腔中取出的动物后,停止电机活动,成为罕见的闪烁速率。
    2. 打下的动物,一个操作站上颈椎脱位并杀死动物。切断从后面肢体的股骨和胫骨,去除皮肤和肌肉。
    3. 70%异丙醇几秒钟解剖股骨和胫骨,并转移到1X的D - PBS。
    4. 在生物安全柜,股骨和胫骨被转移到一个10厘米菜的DMEM。每个骨骼,然后用镊子举行,并用剪刀打开两端被切断。将一个22G的针头到3毫升注射器和填充用DMEM,然后冲入一个50毫升管插入一个开口端的骨针的骨髓。重复2〜3倍,每块骨骼。当所有的骨髓获得,悬浮细胞,并通过一个70μm细胞过滤器的细胞悬液,以去除骨碎片和血液总量。
    5. 离心200克细胞,4℃5分钟,取出吸上清液。于25ml MSC介质的悬浮细胞(DMEM含10%胎牛血清和1%的笔链球菌)。 10ml细胞悬浮液中接种到每一个10cm的培养皿中,共两道菜。 1〜2周保持在37℃和5%的CO 2培养箱培养皿。换液,每隔2〜3天。

    2。富集大鼠MSCs

    已被用于一些表面蛋白,以丰富的干细胞,包括CD54,CD90,CD73,CD105和CD271 3-5 。在我们的研究中,我们作为标记CD54和CD90,以丰富磁性细胞分选干细胞。

    1. 当细胞达到80%左右汇合,吸出培养基,添加4〜5毫升胰蛋白酶EDTA每一道菜。把菜肴的孵化器和孵化约5分钟,以使细胞脱落。一旦细胞分离,加等量的培养基灭活胰蛋白酶。收集到一个15毫升管和自旋细胞,细胞悬液200克,4℃5分钟。
    2. 接下来的步骤描述如何充实两个表面标志物CD54和CD90干细胞,根据细胞分离使用屋宇署IMagnet手册。悬浮细胞沉淀在细胞染色缓冲液(3%热灭活FBS 1X的D - PBS)为20万细胞/ ml。添加生物素标记的CD54抗体(0.25μg每百万个细胞)和生物素标记的CD90抗体(0.15μg每百万个细胞),轻轻混合细胞悬液。冰15分钟的潜伏期后,标记的细胞被洗净,用1X BD IMAG缓冲区的过剩量。标记的细胞被降速200克,4℃5分钟。
    3. 涡屋宇署IMAG链霉亲和素粒子彻底,加40μl颗粒每10万个单元格。彻底标记细胞混合粒子和细胞孵育6〜12℃30分钟。这允许绑定到链霉亲和颗粒的生物素标记的抗CD54和抗CD90,这是必然的表面蛋白CD54和CD90分别。
    4. 在孵化时,标签的圆底试管收集的积极的一小部分。孵化后,将标签卷20万个细胞/毫升1X屋宇署IMAG缓冲区和标记细胞转移到积极的馏分收集管。放置到BD IMagnet的积极的一小部分管,并让它停留6分钟,然后取出上清液用玻璃巴斯德吸管的积极的一小部分管的BD IMagnet仍然。
    5. 屋宇署IMagnet的积极的一小部分管取出,并置于冰上。添加1毫升冰冷1X屋宇署IMAG缓冲区,并轻轻搅拌悬浮细胞。管放置到BD IMagnet并让它留2〜4分钟。取出上清液用一个新的玻璃巴斯德吸管。重复洗涤2.5之一更多的时间。
    6. 删除屋宇署IMagnet和悬浮细胞培养液中,种子之一75厘米2瓶维持细胞和流式细胞仪10厘米菜管。

    3。流式细胞仪检测表面标志表达的验证

    流式细胞仪分析,以验证我们得到的细胞表达CD54和CD90。 HSC的标记CD45的被用来证实,干细胞造血干细胞耗尽。

    1. 当细胞成为〜80%汇合,trypsinize用胰蛋白酶- EDTA的细胞,并收集到一个15毫升管细胞。在200克,4 ° C下5分钟离心收集细胞。
    2. 吸弃上清液,并H细胞与1X的D - PBS一次。悬浮细胞在细胞染色缓冲液(1X的D - PBS含2%FBS和0.05%叠氮钠)的终浓度为5〜10万细胞/毫升,并保持在冰上的细胞。 (; 2亚型控制IgG2a;。3同型对照IgG1的; CD45; 5 CD54的; 6 CD90细胞只)分装100微升到6个标记的试管中的细胞悬液。
    3. 添加适当浓度的同型对照和初级抗体(IgG2a和IgG1:20μL,每百万个细胞; CD45:0.5μg每百万个细胞; CD54:0.25μg每百万个细胞; CD90:0.15μg每百万个细胞),并在4 ° C孵育30分钟。
    4. 1X的D - PBS洗涤细胞两次,然后悬浮细胞100μL细胞染色缓冲。新增的SA - PE(藻红蛋白链霉菌,使用每百万个细胞0.15μg)和孵育4℃30分钟,在黑暗的彗星。
    5. 1X的D - PBS清洗两次标记的细胞。在400μl细胞染色缓冲液重悬细胞,流式细胞仪分析和转让猎鹰管。

    4。衍生的干细胞分离单细胞集落

    干细胞是一种异质性的人口与不同的细胞形态,生长速度以及分化能力6的不同亚群组成。然而,所有的亚群表达称为MSC的标记,因此它是不可行的使用标记分离出这些亚群。因此,我们应用克隆气瓶分离出不同的亚群,这是由单细胞组成的殖民地。

    1. 在大约50〜100个细胞10厘米菜板细胞。在37℃和5%CO 2培养箱中孵育1〜2周。在此期间,研究倒置显微镜以及孤立的殖民地。一旦殖民地已经达到了足够大的尺寸(更好的超过100个细胞在每一个殖民地),马克在盘底部sharpie的殖民地。当挑选标明的殖民地,确保附近捡到一个有没有周围的殖民地。
    2. 吸中期和1X的D - PBS洗一次菜。拿起无菌克隆气缸,用无菌镊子轻轻周围放置标记的殖民地。重复此标记的殖民地,直到所有克隆缸放置在。挑选菌落应远离对方,每一个克隆缸只包含一个殖民地。
    3. 加入100μL胰蛋白酶EDTA每个克隆缸,并把这道菜〜5分钟的孵化器。 5分钟后,细胞在显微镜下检查,看看他们是否四舍五入。当细胞有向上抬起,加等量的培养基灭活胰蛋白酶。用200μL移液器混合细胞悬液,细胞悬液转移到60毫米菜含有3毫升预热培养基。标签正确的菜,放入孵化器。在未来数天的跟踪形态变化。

    5。代表性的成果

    据塑料贴壁细胞在大鼠MSCs隔离的一部分描述的协议,应该是可见的镀后的第二天。当细胞继续增殖,看起来应该像图1A所示的细胞融合细胞。当细胞到达〜80%汇合(图1B),传代,可以进行。在继代培养,胰蛋白酶EDTA用于分离细胞和解除细胞小而圆图1C所示。

    图1
    图1。大鼠MSCs的相衬图像。 (一)聚合MSCS。多数细胞的纺锤状或明星般的。 (二)干细胞在80%左右汇合。 (三)解除后胰蛋白酶的细胞小而圆。

    一旦干细胞富集磁性细胞分选,流式细胞仪分析,以验证表面标志表达。如果浓缩是好的,细胞显示对HSC的标记CD45(图2)对MSC的标志物CD54和CD90阳性染色阴性。同型对照IgG2a和IgG1被用来作为阴性对照。

    图2
    图2。流干细胞表面标志的流式细胞仪分析 。MSCs的标记与IgG2a,IgG1的(2亚型控制)(同型对照1),CD45,CD54和CD90抗体。干细胞表达CD54和CD90,但不CD45的。

    当种子细胞在适当的克隆密度,菌落上升,从单细胞。图3A代表一个单细胞形成一个殖民地。克隆气瓶然后可以用来单独来自殖民地的殖民地和细胞可单独培养。图3B和3C代表从两个独立的殖民地衍生细胞。从殖民地1派生的细胞主轴像(图3B),而从殖民地2所产生的细胞是圆形(图3C)。


    图3。干细胞和单细胞集落来源的细胞集落形成。干细胞培养克隆密度形成单个菌落。这些殖民地可以分离克隆气瓶和细胞从不同的殖民地,可以单独培养。 (A)代表菌落形成干细胞克隆密度电镀时。 (二)梭形细胞派生从一个殖民地。 (三)圆形细胞来自另一个殖民地。

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    Discussion

    本协议描述了如何隔离和丰富的MSCs。一个分开的单细胞集落来源的细胞的方法也纳入其中。有几个步骤,是一个成功的分离,浓缩和殖民地分离的重要。虽然从大鼠细胞分离,这是建议通过细胞过滤器或类似大小的无菌尼龙网,清除血块和骨碎片的过滤器。电镀后的细胞在一夜之间,许多死细胞,将浮在中期和去除死细胞更换新鲜培养基,这应有助于贴壁细胞的生长。

    磁性细胞,在这个协议中的排序描述如何执行积极的选择,并且可以使用类似的程序来执行一个负选择。要添加的抗体量可能会有所不同,需要优化,以达到更好的排序。这也是真正的标记流式细胞仪分析细胞。如果没有运行标记的样品立即流式细胞仪分析,样品可以用2%甲醛固定,并在以后运行。然而,长期储存不建议,因为这会增加自体荧光和牺牲样品质量。

    殖民地分离的关键部分是在合适的细胞密度播种(这应该是优化实验)和定位不是由其他克隆包围的单一克隆。如果附近有其他克隆,克隆缸可能包括附近的克隆,获得的细胞将不再从一个克隆。当放置在克隆的克隆缸,也可以小心滑过盘表面,因为这会导致克隆缸底部硅脂覆盖细胞和深远的细胞分离,防止胰蛋白酶。

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    Disclosures

    Acknowledgements

    这项工作是支持部分由国立卫生研究院(R01GM079688,R21RR024439,R21GM075838),美国国家科学基金会(CBET 0941055)和密歇根州立大学基金会。

    Materials

    Name Company Catalog Number Comments
    1X D-PBS Invitrogen 14040-133
    DMEM Invitrogen 11885-084
    Cell strainer BD Biosciences 352350
    FBS Invitrogen 26140-079
    Pen-Strep Invitrogen 15140
    Trypsin-EDTA Invitrogen 25200-056
    BD Imagnet BD Biosciences 552311
    Biotin mouse IgG2a BD Biosciences 555572
    Biotin mouse IgG1 BD Biosciences 555747
    Biotin anti-rat CD54 Cedarlane Labs CL054B
    Biotin anti-rat CD90 Cedarlane Labs CL005B
    Biotin anti-rat CD45 Cedarlane Labs CL001B
    BD Imag buffer BD Biosciences 552362
    Round-bottom tube BD Biosciences 352063
    Streptavidin particles BD Biosciences 557812
    SA-PE R&D Systems F0040
    Cloning cylinder Sigma-Aldrich C2059

    References

    1. de Hemptinne, I., Vermeiren, C., Maloteaux, J. M., Hermans, E. Induction of glial glutamate transporters in adult mesenchymal stem cells. J Neurochem. 91, 155-166 (2004).
    2. Porter, R. M., Huckle, W. R., Goldstein, A. S. Effect of dexamethasone withdrawal on osteoblastic differentiation of bone marrow stromal cells. J Cell Biochem. 90, 13-22 (2003).
    3. Chamberlain, G., Fox, J., Ashton, B., Middleton, J. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells. 25, 2739-2749 (2007).
    4. Abdallah, B. M., Kassem, M. Human mesenchymal stem cells: from basic biology to clinical applications. Gene Ther. 15, 109-116 (2008).
    5. Erica, L., Herzog, L. C., Diane, S. Krause Plasticity of marrow-derived stem cells. Blood. 102, 3483-3493 (2003).
    6. Colter, D. C., Sekiya, I., Prockop, D. J. Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci U S A. 98, 7841-7845 (2001).

    Comments

    38 Comments

    hi,

    That is a really good explanation about how to derive the mesenchymal stem cells from rats. My work involves derivation of mesenchymal stem cells from mice. I tried to derive them in a similar way but was unsuccessful as my colonies dŒs not have any spindle shaped or round shaped as shown above. So, I would like to ask you can i implement the same kind of steps that are shown above for derivation of MSCs?? Further, how often do i have to change the medium once I plate the primary mesenchymal stem cells derived from the limbs?? You have not mentioned the percentage of trypsin u have used for deattachment of MSCs from the plastic plates?? I have used around 0.²5% trypsin with 5mins of incubation at 37C. But still the cells are not deattaching from the plates.
    Hope to get a reply soon from you.

    regards,
    Chandra Sekhar Amara
    Ph.D. Student
    Hartmann Lab,
    IMP, Vienna, Austria
    Reply

    Posted by: AnonymousMarch 29, 2010, 5:38 PM

    Hi Chandra,

    I haven't worked with mice MSCs and am not very sure whether you can get cells of the same shapes as rat MSCs. But you can try the experimental steps as listed. I usually keep ² dishes for every fresh isolation. Then I change media one day after isolation for one dish, and the other dish I change media two days after isolation. Some times change media one day after is better and some times change media two days after is better. After I pick the better dish, then I usually change media every 3 days.

    I used 0.²5% trypsin plus EDTA. Only trypsin itself dŒs not do a good job in lifting the cells. Supplement EDTA makes cell detachment much faster (usually takes 3~4 minutes for rMSCs I used). The trysin-EDTA I used is from invitrogen with catalogue number ²5²00-056.

    Good luck,

    Linxia
    Reply

    Posted by: AnonymousMarch 29, 2010, 9:00 PM

    Hi Linxia,

    I very much enjoyed your explanation in the vedio. I would like to know after you enrich the MSCs, can you stock them for later use and how many times (passage) i can use from one preparation of MSCs?

    best regards,
    Ava GUO
    Ph.D student
    Hong Kong, China
    Reply

    Posted by: AnonymousJune 23, 2010, 11:57 PM

    Hi Ava,

    Yes, you can freeze the cells in liquid nitrogen and use them later. I usually use the cells within ²0 passages.

    Thanks,
    Linxia
    Reply

    Posted by: AnonymousJune 24, 2010, 9:50 AM

    Hi Linxia,
    I am from china, i guess so are you. lol. I cannot get access to this video. Would you please send me a copy to jianhua_lee@1²6.com?
    Thanks a lot!!
    Lijianhua

    Reply

    Posted by: AnonymousOctober 9, 2011, 11:19 PM

    Hi Jianhua,

    Unfortunately we don't have a video copy either. If you don't access to the written up protocol either, I can send you a copy of that. Sorry couldn't help with the video.

    Linxia
    Reply

    Posted by: AnonymousOctober 10, 2011, 10:02 AM

    Hi linxia
    This is JŒ from montreal, would you like to send aslo a copy of protocols to my email address jŒchaochine@gmail.com. and also if possible some pictures of normal rat MSC, so i can compare their shapes with my cells.
    Reply

    Posted by: AnonymousOctober 27, 2011, 10:32 PM

    Hi JŒ,

    Sure. I will send you a copy of the protocol. As for the pictures, could you check out the figures in the protocol first and see if those are what you are looking for. If you need more, I am willing to send you some other figures we have.

    Linxia
    Reply

    Posted by: AnonymousOctober 28, 2011, 10:49 AM

    Dear Linxia
    I am Kinga from Budapest, Hungary. Could you please send me a copy of your protocol, I am starting my work with rat mesenchymal stem cells now and it would be really helpful.
    lakatoskinga87@gmail.com
    thank you very much
    Kinga Lakatos
    Reply

    Posted by: AnonymousNovember 16, 2011, 4:34 AM

    Hi Kinga,

    Sure, I will send you a copy of the protocol.

    Good luck,
    Linxia
    Reply

    Posted by: AnonymousNovember 16, 2011, 9:47 AM

    Hi LinXia,
    It is such a good video about how to derive the mesenchymal stem cells from rats and seperate the single-colony MSCs. I derive MSCs as shown above. But the cells are not deattaching from the ²5 flask with trypsin plus EDTA which is purchased from invitrogen with catalogue number ²5²00-056 as you describe. If there is any other reason, could you tell me?
    Looking forwad to getting a reply soon from you.

    best regards,
    Lijie Huang
    Ph.D. Student
    Wenzhou Medical College
    Wenzhou, Zhejiang, China
    Reply

    Posted by: AnonymousDecember 13, 2011, 1:00 AM

    Hi Lijie,

    If the cells are not lifting from the surface, try to incubate in Trypsin-EDTA for a little bit longer. After 5min, you can gentlly shake the flask to see whether the cells are coming up. If not, give them a couple of more minutes. Also make sure the Trypsin you get has EDTA in it. If it is only Trypsin (without EDTA), it takes much longer for the cells to deattach. Hope it helps.

    Good luck,
    Linxia
    Reply

    Posted by: AnonymousDecember 13, 2011, 11:55 AM

    Dear Linxia,

    I really need mouse bone marrow-derived messenchymal stem cell for my research but i haven't isolated them yet.
    Although your protocol is used for isolation rat bone marrow-MSC, i think i can use for my work. So can you send me a copy of your protocol to my email address: ntknguyen@hcmus.edu.vn
    Looking forwad to getting a reply soon from you.

    Nguyen Thi Kim Nguyen
    Laboratory of Stem Cell Research & Application
    Viet Nam National university of scinece, Viet Nam
    Reply

    Posted by: Nguyen Thi K.January 7, 2012, 10:02 AM

    Hi Nguyen,

    Just send you a copy of the protocol. Hope it helps.

    Linxia
    Reply

    Posted by: AnonymousJanuary 7, 2012, 10:16 AM

    Hi Linxia,

    I really enjoyed the video. I have isolated cell mesenchymal from murine bone marrow, but cultures do not proliferate (only until the second passages), I have used low-glucose DMEM plus 10% fetal bovine serum and Pen-Strep. Could you help me?


    Thanks,
    Reply

    Posted by: AnonymousJanuary 16, 2012, 11:44 AM

    Hi there,

    I have also experienced bad isolations. Some times the cells proliferate very slowly, show up as very large and flat morphology. These are senescent cells and is very unlikely to make them regrow healthy and fast. When this happens, I would do another isolation to get new cells and usually would get rid of the problem.
    Another thing is don't use medium that's been sitting in the fridge for very long. The pH changes over time and that affect the growth of the cells.

    Hope it helps,

    Linxia
    Reply

    Posted by: AnonymousJanuary 16, 2012, 1:50 PM

    Hi Linxia,
    I cannot get access to this video. I am very interested about MSC subpopulation. What can you tell me about this?Is it possible to have the protocol to separate single-colony derived cells using cloning cylinders ?
    Thank you very much
    mariarosaria
    Reply

    Posted by: AnonymousJanuary 18, 2012, 10:09 AM

    Hi Mariarosaria,

    Just let me know your email address and I can send you a copy of the protocol, which contains isolation of subpopulations.

    Thanks,
    Linxia

    Linxia
    Reply

    Posted by: AnonymousJanuary 18, 2012, 10:27 AM

    Hi Lixia,
    I dont have access to this video in JOVE. Please send me your protocol to my mail id. My mail id is msvnathan@gmail.com.
    Thanking you,
    S. Vaithinathan
    Reply

    Posted by: AnonymousJanuary 18, 2012, 2:40 PM

    Hi Vaithinathan,

    It's sent. Please check your email.

    Linxia
    Reply

    Posted by: AnonymousJanuary 18, 2012, 3:01 PM

    Dear Linxia

    I would like to know whether you add additional growth factors to the medium so to increase the confluency of the cells as i have carried out the experiment but the cells were quite few in number and never increased in number after a certain period of time (after 10 days).

    please kindly provide me the essential steps and precautions used during culturing and the factors required to increase the cell confluency.

    I have used high-glucose DMEM plus 15% fetal bovine serum and Pen-Strep. Could you help me and provide the composition and stimulating factors required for growth

    Thank
    Reply

    Posted by: AnonymousJanuary 18, 2012, 2:46 PM

    Hi Meera,

    I used low-glucose DMEM plus 10% FBS and pen-strep. No additional growth factors were added to the culture medium. The percentage of MSCs from bone marrow is very low, so for the first passage, you won't see many cells. But after a while, the adherent MSCs should grow and populate very fast and form colonies. If your cell number is not enough and they also don't grow fast enough, it might be related to the isolation. I have also experienced bad isolations. This seem to be also related to the animal that's used. When I use another animal, the problem is usually solved. You may try a new isolation. Also, you can optimize your culture medium. I haven't used high-glucose DMEM so I am not sure whether that would affect the growth of the cells. You could try low-glucose DMEM and also different concentration of FBS to see whether it helps.

    Linxia
    Reply

    Posted by: AnonymousJanuary 18, 2012, 3:09 PM

    Dear Linxia

    Thanks for your reply. I want to just discuss the protocol which i follow for the isolation of MSCs from Swiss albino mouse. I dissect out the femur and tibia, which is placed in DMEM (with L-glutamine, 4.5 gm glucose per litre with sodium pyruvate) with Pen/Strep soln in ice cold condition. This is placed as such for half an hour as i would be removing the tissues so to get bones only during flushing. While flushing I transfer the bone segments into Pen/Strp soln and one by one I flush the bone marrow cells with DMEM supplemented with 15 % FBS. When everything has been flushed out with the help of the syringe and the bones have become pale in colour. I centrifuge the whole cells and plate the pellets in DMEM supplemented with FBS. I use a primitive technique for isolation of MSC by plastic adherence. I change this media in 3 hours, maximum cells are aspirated out (HSCs). Then next day firstly I remove the media and allow the cells to grow with removal of media every day.

    Thus by this protocol I get cells which are adherent. I am unable to get these cells into confluent stage, I have studied that certain mesenchymal stimulating factors are available which are added to attain confluency. I studied ur papers abstract but it also mention no such use.

    Could u tell me the concentration of glucose in low-glucose DMEM.

    As I dont have access to this video in JOVE. Please send this protocol to my mail id and please do help me out in this context.

    Thank you so much in advance waiting for ur reply
    Reply

    Posted by: AnonymousJanuary 19, 2012, 12:40 AM

    Hi Meera,

    The DMEM I used is from Invitrogen (Cat # 11885-084). The complete formulation can be found from this website: http://www.invitrogen.com/site/us/en/home/support/Product-Technical-Resources/media_formulation.48.html.
    I see that you change medium 3 hours after plating your cells. Next time when you plating the cells, I would suggest that you plate several dishes. For dish 1, change medium as you normally do (say 3h after plating, or 6h later); for dish ², change medium the next day; for dish 3, change medium ² days later. From my experience, some times change medium the next day gives me better results while other times change medium ² days later gives me better results. I have not tried change medium 3 hours after plating, since I want to give the cells enough time to attach. You could try this out to see whether it may help.

    Also, you are isolating MSCs from mouse, which is also different from isolating from rat. I haven't done isolation from mouse before, so I am not sure if there are other critical steps involved. Maybe you could also look into some literature that particularly describes mouse MSC isolation.

    BTW, I still couldn't see you email address. You can drop me an email at zhanglin@msu.edu and I can send you a copy of the protocol.


    Good luck,
    Linxia
    Reply

    Posted by: AnonymousJanuary 19, 2012, 10:48 AM

    Dear Linxia,

    I am sorry couldn't get ur mail, Please send me your protocol to my mail id. My mail id is meera_nair30@rediffmail.com

    Thanking you,

    Meera
    Reply

    Posted by: AnonymousFebruary 4, 2012, 4:54 AM

    Hi Meera,

    The protocol is sent. Thanks, Linxia
    Reply

    Posted by: AnonymousFebruary 4, 2012, 10:27 AM

    Dear Linxia,
    I wish to know what is the freezing media you are using?
    and if you may, could you tell me the freezing procedure?
    Sincerely,
    Naama
    Reply

    Posted by: AnonymousFebruary 9, 2012, 2:44 AM

    Hi Naama,

    The cryopreservative medium I used is ²0% FBS and ²0% DMSO in MSC culture medium (But the final concentration is 10% FBS and 10% DMSO, please read the following text for why). Here is how I do it: First trypsinize MSCs, then add culture medium to stop trypsinization and collect cells. Spin down the cells and get rid of the supernatant medium. Resuspend cells in MSC culture medium, then add an equal amount of cryopreservative medium slowly and drop-wise. Mix quickly and gently (Now DMSO is 10%). Place cells in vials, put cells at -²0oC for a couple of hours, then put cells at -80oC overnight. The next day, store cells in liquid N². Hope it helps.

    Linxia
    Reply

    Posted by: AnonymousFebruary 9, 2012, 7:18 PM

    Hi, Thanks!!!!
    Reply

    Posted by: AnonymousFebruary 12, 2012, 1:59 AM

    Dear Linxia Zhang,

    Would you send to me your protocol?
    my email is Tung.biotech@gmail.com
    Thank you so much!

    Nguyen Thanh Tung
    Reply

    Posted by: Tung N.June 25, 2012, 3:35 AM

    Hi Nguyen, protocol has been sent. Thanks, Linxia
    Reply

    Posted by: AnonymousJune 25, 2012, 9:34 AM

    Dear Linxia
    I'm also working with rat MSC. I was wondering how you managed to get a good single cell suspension for FACS analysis because my cells like to attach to each other.
    And do you use any kind of positive control for you CD45- cells and isotypes?
    Thank you,
    Fabian
    Reply

    Posted by: Fabian L.January 28, 2013, 8:16 AM

    Hi Fabian,

    Usually my single cell suspension is pretty good. If you keep having problem, you could try use a 50um cell strainer to get rid of cell clumps. The MSCs we get are CD45-CD54+CD90+. I use magnetic cell sorting, flow cytometry is just used to verify that these cells dŒs express CD54 and CD90, but not CD45. I don't think you need a positive control for CD45- and isotypes here. The peaks for the CD45- and isotypes basically overlaps with the 'cells only' control, which dŒs not have fluorescence.

    Thanks,
    Linxia
    Reply

    Posted by: AnonymousJanuary 28, 2013, 10:44 AM

    Hi Linxia,

    thank you for the detailed video. I'm working on RCS rat and isolate MSC from it. I've followed your protocol but got CD45+/CD54+/CD90+ cell population. Would longer incubation (almost ² weeks for each incubation) cause the problem? Would higher concentration of Ab cause the false results? Other than that, I don't know what should be pay attention. Do you have any suggestions? Thanks!

    yuchun
    Reply

    Posted by: yuchun t.April 11, 2013, 8:33 PM

    Hi Yuchun,

    When you say "almost ² weeks for each incubation", do you mean you incubate the cells with the primary antibody for ~² weeks? I only incubate with the primary antibody for ~30min on ice. Also, you probably need to optimize the antibody concentration. Try a couple of dilutions and pick the best one from your FACS results, then stick to that concentration. Hope it helps.

    Thanks,
    Linxia
    Reply

    Posted by: Linxia Z.April 15, 2013, 10:22 AM

    Hi Linxia,

    thanks for your answer. No, the cells are harvested for ² weeks (1st: isolation from femurs and tibias, ²nd: after enrichment iwth CD54, and CD90). For primary antibody I incubate also 30min on ice. It seems like the selection (CD54 & CD90) dŒsn't work. Or other possibilities? Thanks a lot!

    yuchun

    Reply

    Posted by: yuchun t.April 15, 2013, 12:27 PM

    Hi Yuchun,

    So you get CD45+/CD54+/CD90+ cells? Do you do cell sorting before verify with FACS? What you could do is to sort out CD45- cells, then sort out CD54+/CD90+ cells from the CD45- population. Also, make sure your CD45 antibody is good. Some polyclonal antibodies may have lots of non-specific binding. It may recognize other antigens and therefore give you a positive signal. You may want to try with another antibody to see if it fixes the problem.

    Linxia
    Reply

    Posted by: Linxia Z.April 15, 2013, 12:41 PM

    Hi Linxia,

    I would be really very thankful if you have some time to forward the protocol to mariam.foad@gmail.com,

    Thank you,
    Mariam
    Reply

    Posted by: mariam f.February 13, 2014, 8:54 AM

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