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FACS-based Satellite Cell Isolation From Mouse Hind Limb Muscles
基于FACS方法的小鼠后肢肌肉中卫星细胞的分离   

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Abstract

Fluorescence Activated Cell Sorting (FACS) is a sensitive and accurate method for purifying satellite cells, or muscle stem cells, from adult mouse skeletal muscle (Liu et al., 2013; Sacco et al., 2008; Tierney et al., 2014). Mechanical and enzymatic digestion of hind limb muscles releases mononuclear muscle cells into suspension. This protocol employs fractionation strategies to deplete cells expressing the cell surface markers CD45, CD31, CD11b and Ly-6A/E-Sca1, both by magnetic separation and FACS-based exclusion, and positively select for cells expressing a7-integrin and CD34. This enables the researcher to successfully enrich satellite cells that uniformly express the paired-box transcription factor Pax7 and are capable of long-term self-renewal, skeletal muscle repair and muscle stem cell pool repopulation.

Materials and Reagents

  1. Laboratory mice, C57BL/6 strain, 2-4 months of age
    Note: All protocols have been approved by the Sanford-Burnham Medical Research Institute Animal Care and Use Committee.
  2. Isoflurane (Santa Cruz Biotechnology, catalog number: sc-363629Rx )
  3. Ham’s F-10 media (Life Technologies, Gibco®, catalog number: 11550-043 )
  4. Horse serum (Life Technologies, Gibco®, catalog number: 16050-122 )
  5. Collagenase type II (Life Technologies, Gibco®, catalog number: 17101-015 )
  6. Dispase II (Roche Diagnostics, catalog number: 04942078001 )
  7. Phosphate buffered saline (PBS) (pH 7.4) (Life Technologies, catalog number: 10010-023 )
  8. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
  9. Goat serum (Life Technologies, Gibco®, catalog number: 16210-072 )
  10. Antibodies
    1. Biotin CD45 (clone 30-F11, 0.5 mg/ml) (BD Biosciences, catalog number: 553078 )
    2. Biotin CD31 (clone 390, 0.5 mg/ml) (eBioscience, catalog number: 13-0311-85 )
    3. Biotin CD11b (clone M1/70, 0.5 mg/ml) (BD Biosciences, catalog number: 553309 )
    4. Biotin Ly-6A/E-Sca1 (clone E13-161.7, 0.5 mg/ml) (BD Biosciences, catalog number: 553334 )
    5. 7-integrin/PE or FITC (clone R2F2, 1 mg/ml) (AbLabs, catalog number: 53-0010-01 )
    6. CD34/Alexa Fluor 647 (clone RAM34, 0.2 mg/ml) (BD Biosciences, catalog number: 560230 )
    7. Streptavidin APC-Cy7 (0.2 mg/ml) (BD Biosciences, catalog number: 554063 )
    8. Streptavidin microbeads (Miltenyi Biotec, catalog number: 130-048-101 )
    9. FxCycle Violet stain (DAPI, 0.5 mg/ml) (Life Technologies, InvitrogenTM, catalog number: F10347 )
  11. Media (see Recipes)
  12. Stock collagenase type II solution (see Recipes)
  13. Digestion media I (see Recipes)
  14. Digestion media II (see Recipes)
  15. FACS buffer (see Recipes)

Equipment

  1. Isoflurane vaporizer, supply gas (oxygen), flowmeter and induction chamber
  2. TC-treated culture dish (10 cm) (Corning Incorporated, catalog number: 430167 )
  3. Polypropylene centrifuge tubes, sterile (50 ml) (BD Biosciences, catalog number: 352098 )
  4. Polypropylene centrifuge tubes, sterile (15 ml) (BD Biosciences, catalog number: 352096 )
  5. LS columns (Miltenyi Biotec, catalog number: 130-042-401 )
  6. Cell strainers (70 μm) (Thermo Fisher Scientific, catalog number: 22363548 )
  7. Syringes with 20G x 1’’ needles (BD Biosciences, catalog number: 309644 )
  8. 5 ml FACS round-bottom tubes (BD Biosciences, Falcon®, catalog number: 352063 )
  9. Tools for muscle dissection and mincing: Razor blades and/or small scissors, forceps
  10. Tissue culture laminar flow hood
  11. Shaking water bath
  12. Standard temperature-controlled table-top centrifuge
  13. MACS magnetic separator and multi-stand (Miltenyi Biotec, catalog number: 130-042-302 and 130-042-303 )
  14. Flow cytometer [, (FACSAria cell sorter equipped with 488, 405 and 633 nm lasers) (BD Biosciences)
    Note: Color combinations can be adjusted to match the laser combinations available.

Software

  1. FlowJo software (Tree Star, optional)

Procedure

  1. Warm media in 37 °C water bath.
  2. On a surgical bench, harvest the hind limb muscles; place all muscles from one hind limb in a 10 cm dish with 4 ml pre-warmed media (one mouse at a time, keep isolated muscles at 37 °C) (Video 1).
    1. Anesthetize mouse by isoflurane inhalation and sacrifice by cervical dislocation.
    2. Remove skin covering hind limb muscles.
    3. Sever the Achilles tendon and separate the gastrocnemius and soleus.
    4. Sever the distal tendons of the anterior compartment muscles including the tibialis anterior and extensor digitorum longus, use a razor blade to separate from the tibia.
    5. Sever the quadriceps tendon, use razor blade to separate the quadriceps from the femur.


    Video 1. Hind limb muscle isolation

  3. Mince muscles from one hind limb in the 10 cm dish with media using 2 razor blades into small pieces (1-2 plates at a time, keep all other samples at 37 °C).
  4. Once all muscles are minced, use forceps to transfer muscle pieces from each dish into a 15 ml centrifuge tube. Pieces should stick together and be easily picked up as one or two aggregates and placed into the tube (Figures 1 and 2).
    1. Using a 5 ml pipet, collect the media from the dish and any muscle pieces that were not picked up with forceps and add it to the 15 ml centrifuge tube.
    2. Wash the plate with 5 ml media and add it to the 15 ml centrifuge tube. Each tube now contains 9 ml media and the muscle pieces.
    3. Add 1 ml stock collagenase solution to reach a final volume of 10 ml of digestion media I.


    Figure 1. Minced muscle transfer


    Figure 2. Mincing

  5. Place samples on a shaking water bath, warmed to 37 °C, at 175-325 rpm for 90 min. Alternatively, if a shaking water bath is not available, place samples in 37 °C incubator and mix contents manually by inverting the tube every 15-20 min. However, this is likely to reduce yield due to sub-optimal digestion.
  6. Centrifuge samples at 300 x g for 5 min at RT.
  7. Aspirate supernatant and resuspend in 10 ml digestion media II.
  8. Briefly vortex samples and return to the 37 °C shaking water bath at 200 rpm for 30 min.
  9. Pipet up/down to break up muscle pieces and pass each sample through a 10 ml syringe with 20 G needle 10 times to further release all mononuclear cells into suspension. No visible muscle pieces should remain once completed.
  10. Transfer samples to a 50 ml centrifuge tube, washing each 15 ml centrifuge tube 2 times with 12 ml media.
  11. Centrifuge samples at 300 x g for 5 min at 4 °C.
  12. Aspirate supernatant, resuspend samples in 10 ml media and pass through a 70 μm cell strainer atop a fresh 50ml centrifuge tube; wash the original tube 2 times with 12 ml media.
  13. Centrifuge samples at 300 x g for 5 min at 4 °C.
  14. Aspirate supernatant, resuspend samples in 600 μl media and transfer to 15 ml centrifuge tube, then wash the original tube with 400 μl media. Samples from both legs can be combined into one tube at this step such that the sort sample contains cells from all muscles of one mouse in 2 ml media.
  15. Add 10 μl cell suspension from the 2 ml sort sample in 500 μl media and the following volumes of antibody to separate 5 ml FACS tubes for single color controls:
    1. No antibody (unstained control)
    2. 1 μl FxCycle Violet stain (DAPI)
    3. 1 μl α7-integrin/PE or FITC
    4. 4 μl CD34/Alexa Fluor 647
    5. 1 μl biotin CD45
  16. Add the following volumes of primary antibodies to together the original 2 ml sort samples:
    1. 5 μl biotin CD45
    2. 10 μl biotin CD11b
    3. 10 μl biotin CD31
    4. 10 μl biotin Ly-6A/E-Sca1
  17. Gently vortex samples and incubate on ice for 20 min.
  18. Add 10 ml chilled media to sort samples or 3 ml FACS buffer to single color controls.
  19. Centrifuge samples at 300 x g for 5 min at 4 °C.
  20. Aspirate supernatant, resuspend sort samples in 1.5 ml media or single color controls in 500 μl FACS buffer.
  21. Add 1 μl streptavidin APC-Cy7 to the biotin CD45 single color control and incubate on ice for 20 min.
  22. Add the following secondary antibodies to sort samples (microbeads first, then remaining antibodies):
    1. 150 μl streptavidin microbeads
    2. 10 μl α7-integrin/PE or FITC
    3. 30 μl CD34/Alexa Fluor 647
    4. 10 μl streptavidin APC-Cy7
  23. Gently vortex samples and incubate on ice for 20 min.
  24. Add 10 ml chilled media to sort samples or 3 ml FACS buffer to the biotin CD45 single color control.
  25. Centrifuge samples at 300 x g for 5 min at 4 °C.
  26. Pre-load LS columns (one for each sample) for depletion with 3 ml media.
  27. Aspirate supernatant, resuspend sort samples in 1 ml media or the biotin CD45 single color control in 500 μl FACS buffer.
  28. Load each sort sample onto an LS column placed on a MACS magnetic separator, washing the original centrifuge tube 2 times with 3-4 ml media (keeping samples on ice as much as possible).
  29. Centrifuge samples at 300 x g for 5 min at 4 °C.
  30. Aspirate supernatant, resuspend sort samples in 2 ml FACS buffer transferred to 5 ml FACS tubes and add 2 μl FxCycle Violet stain (DAPI) to each sample.
  31. Keep on ice until ready for sorting on a BD Biosciences FACSAria cell sorter (Figure 3).
    a. Cell should be sorted at 20 psi through a 100 μm nozzle.


    Figure 3. FACS-based satellite cell isolation. Representative FACS plots and gating schemes to purify mononucleated satellite cells both negative for CD45, CD31, CD11b and Sca1 and expressing a7-integrin and CD34 (left). Representative FACS plot showing a7-integrin and CD34 expression in re-sorted satellite cells.

Notes

  1. During the muscle dissection and mincing steps, look for and discard large pieces of thick white tendons that do not efficiently get digested and may clog the syringe and needle at step 9.
  2. Razor-based mincing prior to enzymatic digestion is a critical step in the protocol; mincing into large pieces will result in an incomplete digestion of the tissue while mincing into small pieces will lower yield.
  3. Supernatant from step 7 can be collected, diluted further with cold PBS, centrifuged at 400 x g for 8 min and kept on ice until added back to main sample prior to filtering at step 12 to potentially increase yield.
  4. Cell suspensions following tissue digestion should be kept on ice or at 4 °C to slow satellite cell activation and sorted as quickly as possible following sample preparation, particularly if analysis is planned for freshly isolated cells when presumed quiescent.
  5. FxCycle Violet Stain (DAPI) can be used at 300-900 μg/ml before increased autofluorescence is observed. Alternatively, propidium iodide can be used to discriminate between live and dead cells but fluorescence compensation is more difficult with this color scheme.
  6. Cellular yield from one adult mouse, C57BL/6 strain, 2-4 months of age should be approximately 150,000-200,000 events by FACS analysis and 75,000-100,000 satellite cells when manually counted.

Recipes

  1. Media
    Ham’s F-10 media and 10% horse serum
    0.2 μm sterile filtered and stored at 4 °C
  2. Stock collagenase type II solution
    Weigh out and dissolve collagenase powder in media (Recipe 1) for a stock concentration of 7,000 units/ml
    Units per milligram vary by lot and calculations need to be adjusted accordingly
    Stock collagenase solution can be aliquoted and stored at -20 °C for up to 6 months or 4 °C for 1 day
    Do not freeze/thaw more than one time
  3. Digestion media I
    9 ml media (Recipe 1) and 1 ml stock collagenase type II solution (Recipe 2)
  4. Digestion media II
    9.86 ml media (Recipe 1)
    143 μl stock collagenase type II solution (Recipe 2, 100 units/ml final concentration)
    20 units dispase II (powdered form, weighed out immediately prior to use)
  5. FACS buffer
    486.5 ml 1x PBS
    12.5 ml goat serum (2.5% final) and 1 ml 0.5 M EDTA (1 mM final)
    0.2 μm sterile filtered and stored at 4 °C

Acknowledgments

This protocol was adapted and modified from previous work published by both Tom Rando and Helen Blau’s research groups. This work was supported by the Muscular Dystrophy Association MDA Grant 200845, Ellison Medical Foundation New Scholar Award AG-NS-0843-11 and US National Institutes of Health (NIH) grants R01AR064873, R03 AR063328, P30 AR061303 to AS, and US National Institutes of Health (NIH) grant F31 AR065923-01 to MT.

References

  1. Liu, L., Cheung, T. H., Charville, G. W., Hurgo, B. M., Leavitt, T., Shih, J., Brunet, A. and Rando, T. A. (2013). Chromatin modifications as determinants of muscle stem cell quiescence and chronological aging. Cell Rep 4(1): 189-204.
  2. Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S. and Blau, H. M. (2008). Self-renewal and expansion of single transplanted muscle stem cells. Nature 456(7221): 502-506.
  3. Tierney, M. T., Aydogdu, T., Sala, D., Malecova, B., Gatto, S., Puri, P. L., Latella, L. and Sacco, A. (2014). STAT3 signaling controls satellite cell expansion and skeletal muscle repair. Nat Med 20(10): 1182-1186.

简介

荧光活化细胞分选(FACS)是用于从成年小鼠骨骼肌纯化卫星细胞或肌肉干细胞的灵敏和精确的方法(Liu等人,2013; Sacco等人, ,2008; Tierney ,,2014)。 后肢肌肉的机械和酶消化将单核肌细胞释放到悬浮液中。 该方案采用分级分离策略,通过磁性分离和基于FACS的排除,耗尽表达细胞表面标记CD45,CD31,CD11b和Ly-6A/E-Sca1的细胞,并阳性选择表达α7-整联蛋白和CD34的细胞。 这使得研究者能够成功地富集均匀表达配对盒转录因子Pax7并且能够长期自我更新,骨骼肌修复和肌肉干细胞池再增殖的卫星细胞。

材料和试剂

  1. 实验室小鼠,C57BL/6株,2-4个月龄
    注意:所有协议均已获得Sanford-Burnham医学研究所动物护理和使用委员会批准。
  2. 异氟烷(Santa Cruz Biotechnology,目录号:sc-363629Rx)
  3. Ham's F-10培养基(Life Technologies,Gibco ,目录号:11550-043)
  4. 马血清(Life Technologies,Gibco ,目录号:16050-122)
  5. II型胶原酶(Life Technologies,Gibco ,目录号:17101-015)
  6. Dispase II(Roche Diagnostics,目录号:04942078001)
  7. 磷酸盐缓冲盐水(PBS)(pH 7.4)(Life Technologies,目录号:10010-023)
  8. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E6758)
  9. 山羊血清(Life Technologies,Gibco ,目录号:16210-072)
  10. 抗体
    1. 生物素CD45(克隆30-F11,0.5mg/ml)(BD Biosciences,目录号:553078)
    2. 生物素CD31(克隆390,0.5mg/ml)(eBioscience,目录号:13-0311-85)
    3. 生物素CD11b(克隆M1/70,0.5mg/ml)(BD Biosciences,目录号:553309)
    4. 生物素Ly-6A/E-Sca1(克隆E13-161.7,0.5mg/ml)(BD Biosciences,目录号:553334)
    5. 7-整联蛋白/PE或FITC(克隆R2F2,1mg/ml)(AbLabs,目录号:53-0010-01)
    6. CD34/Alexa Fluor 647(克隆RAM34,0.2mg/ml)(BD Biosciences,目录号:560230)
    7. 链霉亲和素APC-Cy7(0.2mg/ml)(BD Biosciences,目录号:554063)
    8. 链霉亲和素微珠(Miltenyi Biotec,目录号:130-048-101)
    9. FxCycle紫染色剂(DAPI,0.5mg/ml)(Life Technologies,Invitrogen ,目录号:F10347)
  11. 媒体(见配方)
  12. 股票胶原酶II型溶液(见配方)
  13. 消化培养基I(参见食谱)
  14. 消化培养基II(参见食谱)
  15. FACS缓冲液(见配方)

设备

  1. 异氟烷蒸发器,供应气体(氧气),流量计和感应室
  2. TC处理的培养皿(10cm)(Corning Incorporated,目录号:430167)
  3. 无菌(50ml)聚丙烯离心管(BD Biosciences,目录号:352098)
  4. 无菌(15ml)聚丙烯离心管(BD Biosciences,目录号:352096)
  5. LS柱(Miltenyi Biotec,目录号:130-042-401)
  6. 细胞过滤器(70μm)(Thermo Fisher Scientific,目录号:22363548)
  7. 使用20G×1"针(BD Biosciences,目录号:309644)的注射器
  8. 将5ml FACS圆底管(BD Biosciences,Falcon ,目录号:352063)
  9. 用于肌肉解剖和绞碎的工具:剃刀刀片和/或小剪刀,镊子
  10. 组织培养层流罩
  11. 摇水浴
  12. 标准温度控制台式离心机
  13. MACS磁选机和多支架(Miltenyi Biotec,目录号:130-042-302和130-042-303)
  14. 流式细胞仪[,(装备有488,405和633nm激光的FACSAria细胞分选仪)(BD Biosciences)
    注意:可以调整颜色组合以符合可用的激光组合。

软件

  1. FlowJo软件(Tree Star,可选)

程序

  1. 温热介质在37℃水浴中。
  2. 在外科手术台上,收获后肢肌肉; 将来自一个后肢的所有肌肉置于具有4ml预热的培养基(每次一只小鼠,将分离的肌肉保持在37℃)的10cm培养皿中(视频1)。
    1. 通过异氟烷吸入麻醉小鼠,并通过颈椎脱臼处死
    2. 去除覆盖后肢肌肉的皮肤。
    3. 切断跟腱,分离腓肠肌和比目鱼肌。
    4. 切断前房肌肉的远端肌腱,包括 胫骨前伸肌和伸肌伸肌,使用剃刀刀片 从胫骨分离
    5. 分割四头肌腱,使用剃刀刀片将股四头肌与股骨分离。

    视频1.后肢肌肉隔离
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  3. 剁碎肌肉从一个后肢在10厘米的菜,用介质使用2个剃刀刀片成小块(1-2板一次,保持所有其他样品在37℃)。
  4. 一旦所有肌肉被切碎,使用镊子将肌肉片从每个盘转移到15毫升离心管。物件应该粘在一起并且容易地作为一个或两个聚集体拾取并放入管中(图1和图2)。
    1. 使用5毫升吸管,从菜和任何肌肉收集媒体 不用镊子拿起的片断,并将其添加到15毫升 离心管
    2. 用5 ml培养基洗板,加入 15 ml离心管。 每个管现在包含9ml培养基和肌肉 件。
    3. 加入1ml原胶原酶溶液,以达到10ml的消化培养基I的最终体积。


    图1.肌肉转移


    图2.停止

  5. 将样品放在摇动的水浴上,温热至37℃,在175-325rpm下90分钟。 或者,如果摇动水浴不可用,将样品放在37℃培养箱中,通过每15-20分钟倒转管手动混合内容物。 然而,这可能由于次优消化而降低产量。
  6. 在室温下以300×g离心样品5分钟
  7. 吸出上清液并重悬于10ml消化培养基II中
  8. 短暂涡流样品,并返回到37℃摇动水浴,在200rpm下30分钟
  9. 向上/向下移动以破碎肌肉块,并使每个样品通过具有20G针的10ml注射器10次,以进一步释放所有单核细胞成悬浮液。 一旦完成,就不会有可见的肌肉块。
  10. 将样品转移到50ml离心管中,用12ml培养基洗涤每个15ml离心管2次
  11. 在4℃下以300×g离心样品5分钟
  12. 吸出上清液,将样品重悬于10ml培养基中,并通过新鲜50ml离心管顶部的70μm细胞滤器; 用12 ml培养基洗涤原管2次
  13. 在4℃下以300×g离心样品5分钟
  14. 吸出上清液,重悬样品在600μl培养基,并转移到15ml离心管,然后用400μl培养基洗涤原管。 在这一步骤中,可以将来自两条腿的样品合并成一个管,使得分选样品包含来自一只小鼠的所有肌肉的细胞在2ml培养基中。
  15. 加入10微升细胞悬浮液从2毫升分选样品在500微升培养基和以下体积的抗体分开的5毫米FACS管单色控制:
    1. 无抗体(未染色对照)
    2. 1μlFxCycle紫色染料(DAPI)
    3. 1μlα7-整合素/PE或FITC
    4. 4μlCD34/Alexa Fluor 647
    5. 1μl生物素CD45
  16. 将以下体积的一抗添加到原始2ml分选样品中:
    1. 5μl生物素CD45
    2. 10μl生物素CD11b
    3. 10μl生物素CD31
    4. 10μl生物素Ly-6A/E-Sca1
  17. 轻轻涡旋样品,在冰上孵育20分钟
  18. 加入10ml冷冻培养基以分选样品或3ml FACS缓冲液至单色对照
  19. 在4℃下以300×g离心样品5分钟
  20. 吸出上清液,重悬样品在1.5毫升培养基或单色控件在500微升FACS缓冲液
  21. 加入1微升链霉亲和素APC-Cy7到生物素CD45单一颜色控件,并在冰上孵育20分钟。
  22. 添加以下二级抗体以分选样品(首先是微珠,然后是剩余的抗体):
    1. 150μl链霉亲和素微珠
    2. 10μlα7-整合素/PE或FITC
    3. 30μlCD34/Alexa Fluor 647
    4. 10μl链霉亲和素APC-Cy7
  23. 轻轻涡旋样品,在冰上孵育20分钟
  24. 加入10ml冷冻培养基以将样品分选或3ml FACS缓冲液加入生物素CD45单色对照
  25. 在4℃下以300×g离心样品5分钟
  26. 预装LS柱(每个样品一个),用3ml培养基消耗
  27. 吸出上清液,将样品重悬在1ml培养基中,或将生物素CD45单色对照在500μlFACS缓冲液中重悬。
  28. 将每个样品装载到放置在MACS磁性分离器上的LS柱上,用3-4ml培养基(尽可能多地将样品保持在冰上)清洗原离心管2次。
  29. 在4℃下以300×g离心样品5分钟
  30. 吸出上清液,重悬样品在2毫升FACS缓冲液转移到5毫升FACS管,并添加2微升FxCycle紫染料(DAPI)到每个样品。
  31. 保持在冰上,直到准备好在BD Biosciences FACSAria细胞分选仪上分选(图3) 一个。电池应通过100μm喷嘴在20 psi下分类

    图3.基于FACS的卫星细胞分离。代表性的FACS图和门控方案,用于纯化对CD45,CD31,CD11b和Sca1阴性并表达α7-整联蛋白和CD34的单核卫星细胞(左图)。代表FACS图,其显示在重排的卫星细胞中的α7-整联蛋白和CD34表达

笔记

  1. 在肌肉解剖和绞碎步骤期间,寻找并丢弃没有有效消化的大块厚白色腱,并且可能在步骤9堵塞注射器和针。
  2. 在酶消化之前基于剃刀的切碎是方案中的关键步骤;切成大块将导致组织不完全消化,而切成小块将降低产量
  3. 可以收集来自步骤7的上清液,进一步用冷PBS稀释,在400×g离心8分钟并保持在冰上,直到在步骤12过滤之前回到主样品,以潜在地增加产量。 br />
  4. 组织消化后的细胞悬液应保存在冰上或4℃,以减缓卫星细胞活化,并在样品制备后尽可能快地分选,特别是如果在假定静止时对新鲜分离的细胞进行分析。
  5. 在观察到增加的自发荧光之前,可以使用300-900μg/ml的FxCycle紫色染料(DAPI)。 或者,碘化丙啶可用于区分活细胞和死细胞,但是用这种配色方案更难以进行荧光补偿。
  6. 通过FACS分析,来自一个成年小鼠,C57BL/6株,2-4个月龄的细胞产量应当为约150,000-200,000个事件,并且在手动计数时为75,000-100,000个卫星细胞。

食谱

  1. 媒体
    Ham's F-10培养基和10%马血清
    0.2μm无菌过滤并在4℃下保存
  2. 股票胶原酶II型溶液
    称量和溶解胶原酶粉末在介质(配方1)中,原料浓度为7,000单位/ml
    单位每毫克根据批次不同,计算需要相应调整
    可以将股胶原酶溶液等分并在-20℃保存长达6个月或在4℃保存1天。
    不要冻结/解冻多次
  3. 消化培养基我
    9ml培养基(配方1)和1ml原胶原酶II型溶液(配方2)
  4. 消化培养基II
    9.86ml介质(配方1)
    143μl原液胶原酶II型溶液(配方2,100单位/ml终浓度)
    20单位分散II(粉末形式,在使用前称重)
  5. FACS缓冲区
    486.5 ml 1x PBS
    12.5ml山羊血清(2.5%终浓度)和1ml 0.5M EDTA(1mM终浓度) 0.2μm无菌过滤并在4℃下保存

致谢

这个协议是从Tom Rando和Helen Blau的研究小组发布的以前的工作改编和修改的。该工作由肌营养不良症协会MDA Grant 200845,Ellison Medical Foundation新学者奖AG-NS-0843-11和美国国立卫生研究院(NIH)授予R01AR064873,R03 AR063328,P30 AR061303至AS和美国国立研究所健康(NIH)授予F31 AR065923-01至MT。

参考文献

  1. Liu,L.,Cheung,T. H.,Charville,G. W.,Hurgo,B. M.,Leavitt,T.,Shih,J.,Brunet,A.and Rando,T.A。 染色质修饰作为肌肉干细胞静止和时间衰老的决定因素细胞Rep 4(1):189-204。
  2. Sacco,A.,Doyonnas,R.,Kraft,P.,Vitorovic,S。和Blau,H.M。(2008)。 单一移植肌肉干细胞的自我更新和扩增 /em> 456(7221):502-506。
  3. Tierney,M.T.,Aydogdu,T.,Sala,D.,Malecova,B.,Gatto,S.,Puri,P.L.,Latella,L.and Sacco,A。(2014)。 STAT3信号控制卫星细胞扩增和骨骼肌修复。 Nat Med 20(10):1182-1186。
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引用:Gromova, A., Tierney, M. T. and Sacco, A. (2015). FACS-based Satellite Cell Isolation From Mouse Hind Limb Muscles. Bio-protocol 5(16): e1558. DOI: 10.21769/BioProtoc.1558.
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