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Isolation of FAP Cells from Mouse Dystrophic Skeletal Muscle Using Fluorescence Activated Cell Sorting

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Stem Cells
Apr 2014



A population of muscle resident CD45-, CD31- cells expressing the mesenchymal PDGF receptor alpha (PDGFRα) as well as Sca-1 was first isolated in healthy mouse muscles in Uezumi et al. (2010). In the same year, Joe et al. (2010) identified and purified fibro-adipogenic precursors (FAPs), cells located into the interstitial space between myofibers close to vessels, negative for CD45, CD31,α7-Integrin, but expressing CD34, Sca-1.

Both groups demonstrated that these cells are not myogenic in vitro or in vivo, but they are capable of differentiating in vitro towards both fibrogenic and adipogenic lineage (Uezumi et al., 2011). Further marker analysis indicates that the two groups identified independently the same cell population (Natarajan et al., 2010).

FAPs are an important source of fibrosis and adipogenesis in dystrophic skeletal muscle (Natarajan et al., 2010; Cordani et al., 2014). We have recently demonstrated that Nitric Oxide regulates FAP fate inhibiting in vitro their differentiation into adipocytes. In mdx mice, an animal model of DMD, fed with a diet containing the nitric oxide donating drug, Molsidomine, the number of PDGFRα+ cells was reduced as well as the deposition of both skeletal muscle fat and connective tissues (Cordani et al., 2014). Here we described a method to isolate in both wild type and in mdx dystrophic muscle pure population of FAPs by double selection for SCA-1 and PDGFRα positivity in absence of the satellite cell markers SM/C2.6 and α7integrin as well of the pan-lymphocytes marker CD45 or endothelial marker CD31.

Keywords: Fibroadypose precursors (fibroadypose前兆), Skeletal muscle (骨骼肌), Fluorescence cell sorting (荧光细胞分选), Mouse dystrophy model (营养不良模型小鼠), Satellite cells (卫星细胞)

Material and Reagents

  1. 8-10 weeks old mice C57BL/6J wild-type mice (Charles River Laboratories International, http://www.criver.com) and mdx-4cv mice (B6Ros.Cg-Dmdmdx-4cv/J, crossed on C57/BL/6 background; Jackson ImmunoResearch Laboratories)
    Note: Animals were treated in accordance with European Community guidelines and with the approval of the Institutional Ethical Committee.
  2. Collagenase II (Worthington Biochemical, catalog number: CLSS2 )
  3. Dulbecco's modified high glucose Eagle's medium (DMEM high glucose) (EuroClone, catalog number: ECB7501L )
  4. 100 U/ml penicillin and 100 μg/ml streptomycin (EuroClone)
  5. L-glutammine (EuroClone)
  6. Recombinant human basic fibroblast growth factor (b-FGF) (Pepro Tech, catalog number: 100-18B )
  7. Growth factor reduced BD MatrigelTM Matrix (BD Biosciences, catalog number: 356230 )
  8. Foetal Bovine Serum (FBS) (EuroClone)
  9. Sterile phosphate buffered saline (PBS) w/o Ca++Mg++ (EuroClone)
  10. Antibodies
    1. Anti-CD31-phycoerythrin/Cy7 (anti-CD31-PE/Cy7, clone 390) (eBioscience, catalog number: 25-0311 )
    2. Anti-CD45-PECy7 (clone 30-F11) (eBioscience, catalog number: 15-0451 )
    3. Anti-SM/C2.6-Biotin (kindly provided by Dr. Fukada) (Fukada et al., 2004)
    4. Streptavidin-PE (BioLegend)
    5. Anti-α7-Integrin-PE (clone R2F2) (AbLab Laboratorio di Istologia e Citologia Patologica Veterinaria, catalog number: AB10RS24MW215 )
    6. Anti-PDGFRα-allophycocyanin (APC, CD140a, clone APA5) (BioLegend, catalog number: 135907 )
    7. Anti-LY-6A/E SCA-1-allophycocyanin/Cy7 (APC/Cy7, clone B7) (BD Biosciences, catalog number: 560654 )
    8. 7-aminoactinomycin D (7-AAD) (Life Technologies, catalog number: A1310 )
  11. Growth medium (GM) (see Recipes)
  12. Wash buffer (WB) (see Recipes)
  13. Collagenase II solution (see Recipes)
  14. Erythrocytes lysis buffer (see Recipes)
    Note: Use 1 ml of this solution for approximately 1 g of muscle.
  15. Sorting buffer (see Recipes)
  16. Matrigel solution (see Recipes)
  17. b-FGF solution (see Recipes)


  1. Scissors and tweezers
  2. Cell culture plastic dishes (Corning, Costar®)
  3. Six multiwells (Corning, Costar®)
  4. Centrifuge
  5. 50 and 15 ml plastic tubes
  6. 18G-10 ml syringes
  7. 70 μm and 40 μm cell strainer caps (BD Biosciences)
  8. Beckam Coulter Cell Sorter MoFloTM XDP (Beckman Coulter, catalog number: ML99030 )
  9. Cell culture incubator at 37 °C and 5% CO2
  10. Microscope or cell counter


  1. Sacrifice the mice (C57BL/6 or MDX) by delivering increasing concentrations of CO2 and remove hindlimb muscles.
  2. Weigh the muscle mass.
  3. Leave them in cold PBS-containing dish.
  4. Remove visible tendon, adipose tissue and vessel.
  5. Mince the muscles on new dish (not containing PBS) using a curved tip scissor for few minutes until the tissue appears like a mush.
  6. Transfer the tissue into a 50 ml plastic tube and add 0.2% collagenase II in DMEM (serum-free). Volume to use is 2-4 ml of collagenase for 1 g of muscle weight: Usually each mouse allows obtaining 1-1.5 g of tissue. Put plastic tubes into a shaking thermostatic bath, at 37 °C for 1 h.
  7. Separate undigested from digested material by centrifugation at 500 rpm. All the centrifugations are at room temperature. Supernatant (digested material) was removed, diluted with room temperature PBS (approximately 50 ml of PBS/1 g muscle) and go through a 18 G-10 ml syringe to create a single cell suspension (about 10 passages through the needle).
  8. Add to the un-digesting muscle fresh collagenase solution (using the same volume of step 6) and leave in the shaking thermostatic bath, at 37 °C for additional 30 min. Then, repeat step 7. Discard eventually indigested material that may remain after the second centrifugation procedure.
  9. Muscle slurries obtained by the two digestions were pulled and filtered through 70 μm cell strainer cap and subsequently through 40 μm cell strainer cap, using 50 ml syringes and 50 ml plastic tubes.
  10. Centrifuge at 2,000 rpm for 10 min and remove supernatant using a pipette.
  11. Eliminate blood red cells by re-suspending cell with 1-2 ml of erythrocyte lysis buffer.
  12. Dilute erythrocyte lysis buffer by addiction of 50 ml of PBS containing 2% FBS and centrifuge 2,000 rpm for 10 min at room temperature.
  13. After removing the supernatant, using a pipette, add 1 ml PBS containing 10% of FBS and count the cells.
  14. Centrifuge tubes 2,000 rpm for 10 min and incubate recovered cells for 30 min with anti-SM/C2.6-Biotin on ice. Use 1 µl of antibodies for 1 x 106 cells in 100 µl of PBS containing 2% of FBS.
  15. Add 1 ml wash buffer and centrifuge 2,000 rpm for 5 min.
  16. After removing the supernatant, incubate on ice for 30 min with Streptavidin-PE and with the other labeled antibodies [anti-CD31-PECy7 and anti-CD45-PECy7, anti-α7-Integrin-PE (for MDX mice), anti-PDGFRα-APC, anti-LY-6A/E SCA-1-APC/Cy7] and with 7-aminoactinomycin D. As in step 14, use 1 µl of antibodies for 1 x 106 cells in 100 µl of PBS containing 2% of FBS.
  17. Add 50 ml of wash buffer and centrifuge 2,000 rpm for 5 min.
  18. After removing the supernatant, add 2 ml of sorting buffer/mice.
  19. To set up the instrument, sample of about 50-100,000 cells were in parallel individually stained with the each single antibody or with 7-aminoactinomycin (single staining for compensation) in the same conditions of samples to sort. A sample of 50-100,000 cells was also no incubated with any antibody (unstained control). After 30 min, add 1 ml of wash buffer then centrifuge (2,000 rpm for 5 min). Remove supernatant and add 200-300 µl of PBS containing 2% of FBS.
  20. For cell sorter setting: Using unstained control, exclude debris and dead cells by forward scatter and side scatter and set basal fluorescence (Figure 1A). Then make 7-AAD gating using sample stained with 7-aminoactinomycin (gate 1, 7AAD negative cells and gate 2) to further eliminate dead ells.
  21. Compensate fluorescence using the samples individually stained with each antibodies and then select in sample to sort FAP cells as CD45-/CD31- (gate 3)/smc2.6- or α7integrin- (gate 4)/SCA-1+ and PDGFRα+ (gate 5) cells. To improve purity, we preferred to employ anti- smc2.6 for WT mice and anti-α7integrin for mdx mice.
  22. At the end of sorting, plate FAP cells at 1 x 104 cells/cm2 in multiwell plates previously coated with Matrigel and culture for 6 days in growth medium plus b-FGF in a humidified incubator (37 °C and 5% CO2).

Representative data

Figure 1. Cell sorter procedure. A. single cells gating; B. viable cells gating based on selection of 7ADD negative cells (1, left) and forward scatter/side scatter parametes (2, right); C. CD31 and CD45 negative cells gating (3); D. α7-integrin gating (4); E. satellite cells gating (Sca 1/PDGFRα negative cells from α7-integrin positive gate); F. FAP cells gatining (ScA 1/PDGFRα positive cells from the α7-intergrin negative gate (5).


  1. FAP cells recovery range from 1 to 3% of total cells. Digestion seems to be the critical point.


  1. Growth medium (GM)
    Dulbecco's modified high glucose Eagle's medium (DMEM) supplemented with heat inactivated 20% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin plus 5 ng/ml of recombinant human basic fibroblast growth factor
  2. Wash buffer (WB)
    PBS w/o Ca++ and Mg++ with heat inactivated 2% FBS
  3. Collagenase II solution
    0.2% collagenase solution was prepared in DMEM, filtered on a 0.2 µm filter and immediately used.
  4. Erythrocytes lysis buffer
    0.8% NH4Cl in Tris-buffer (pH 8)
  5. Sorting buffer
    PBS w/o Ca2+ and Mg2+ with 5% heat inactivated FBS
  6. Matrigel solution
    Dilute Matrigel 1:100 in DMEM and use immediately after preparation for plate coating
    Left Matrigel working solution into the plate for 30 min before removing
    Matrigel must not dry
  7. b-FGF solution
    Prepare 100 µg/ml stock solution aliquots and keep them at 20 °C
    In culture b-FGF concentration was 5 ng/ml
    After defrosting, b-FGF aliquot could remain at 4 °C and used within a week


We are grateful to Prof. So-ichiro Fukada (Osaka University, Osaka, Japan) for the SMC/2.6 antibody. This work was supported by the European Community’s framework program FP7/2007-2013 under grant agreement n° 241440 (ENDOSTEM), the Italian Ministry of Health RC 2013, Associazione Italiana Ricerca sul Cancro (AIRC IG11362) and from the Ministero della Università e Ricerca PRIN 2010-2011.


  1. Cordani, N., Pisa, V., Pozzi, L., Sciorati, C. and Clementi, E. (2014). Nitric oxide controls fat deposition in dystrophic skeletal muscle by regulating fibro-adipogenic precursor differentiation. Stem Cells 32(4): 874-885.
  2. Fukada, S., Higuchi, S., Segawa, M., Koda, K., Yamamoto, Y., Tsujikawa, K., Kohama, Y., Uezumi, A., Imamura, M., Miyagoe-Suzuki, Y., Takeda, S. and Yamamoto, H. (2004). Purification and cell-surface marker characterization of quiescent satellite cells from murine skeletal muscle by a novel monoclonal antibody. Exp Cell Res 296(2): 245-255.
  3. Joe, A. W., Yi, L., Natarajan, A., Le Grand, F., So, L., Wang, J., Rudnicki, M. A. and Rossi, F. M. (2010). Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis. Nat Cell Biol 12(2): 153-163.
  4. Natarajan, A., Lemos, D. R. and Rossi, F. M. (2010). Fibro/adipogenic progenitors: a double-edged sword in skeletal muscle regeneration. Cell Cycle 9(11): 2045-2046.
  5. Uezumi, A., Fukada, S., Yamamoto, N., Takeda, S. and Tsuchida, K. (2010). Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle. Nat Cell Biol 12(2): 143-152.
  6. Uezumi, A., Ito, T., Morikawa, D., Shimizu, N., Yoneda, T., Segawa, M., Yamaguchi, M., Ogawa, R., Matev, M. M., Miyagoe-Suzuki, Y., Takeda, S., Tsujikawa, K., Tsuchida, K., Yamamoto, H. and Fukada, S. (2011). Fibrosis and adipogenesis originate from a common mesenchymal progenitor in skeletal muscle. J Cell Sci 124(Pt 21): 3654-3664.


首先在Uezumi等人的健康小鼠肌肉中分离表达间充质PDGF受体α(PDGFRα)以及Sca-1的肌肉驻留CD45 - ,CD31 -/- em。等人(2010)。在同一年,Joe等人(2010)鉴定并纯化了成纤维脂肪生成前体(FAP),位于靠近血管的肌纤维之间的间隙空间中的细胞,CD45,CD31,α7-整合素,但是表达CD34,Sca-1。两个小组都证明这些细胞在体外不是肌原性的或在体内,但它们能够区分< (Uezumi等人,2011),其在体外对于纤维形成和脂肪形成谱系起作用。进一步的标记分析表明两组独立地鉴定了相同的细胞群体(Natarajan等人,2010)。
FAP是营养不良骨骼肌中的纤维化和脂肪形成的重要来源(Natarajan& et al。,2010; Cordani et al。,,2014)。我们最近已经证明,一氧化氮调节FAP命运在体外抑制它们向脂肪细胞的分化。在mdx 小鼠中,DMD的动物模型,其中饲喂含有一氧化氮给药药物莫西沙星,PDGFRα +细胞数目的DMD,以及沉积的骨骼肌脂肪和结缔组织(Cordani等人,2014)。在这里我们描述了通过双重选择SCA-1和PDGFRα阳性在不存在卫星细胞标记物SM/C2.6和α7整联蛋白的情况下分离野生型和mdx营养不良肌肉纯FAP群体的方法,淋巴细胞标记物CD45或内皮标记物CD31

关键字:fibroadypose前兆, 骨骼肌, 荧光细胞分选, 营养不良模型小鼠, 卫星细胞


  1. 将8-10周龄小鼠C57BL/6J野生型小鼠(Charles River Laboratories International,http://www.criver.com)和mdx-4cv小鼠(B6Ros.Cg-Dmdmdx-4cv/J,在C57/BL/6 background; Jackson ImmunoResearch Laboratories)
  2. 胶原酶II(Worthington Biochemical,目录号:CLSS2)
  3. Dulbecco改良的高葡萄糖Eagle培养基(DMEM高葡萄糖)(EuroClone,目录号:ECB7501L)
  4. 100U/ml青霉素和100μg/ml链霉素(EuroClone)
  5. L-谷氨酸(EuroClone)
  6. 重组人碱性成纤维细胞生长因子(b-FGF)(Pepro Tech,目录号:100-18B)
  7. 生长因子减少的BD Matrigel Matrix(BD Biosciences,目录号:356230)
  8. 胎牛血清(FBS)(EuroClone)
  9. 无菌磷酸盐缓冲盐水(PBS)w/o Ca ++ ++ Mg ++ (EuroClone)
  10. 抗体
    1. 抗CD31-藻红蛋白/Cy7(抗CD31-PE/Cy7,克隆390)(eBioscience,目录号:25-0311)
    2. 抗CD45-PECy7(克隆30-F11)(eBioscience,目录号:15-0451)
    3. 抗SM/C2.6-生物素(由Fukada博士友情提供)(Fukada等人,2004)
    4. 链霉亲和素-PE(BioLegend)
    5. 抗α7整联蛋白-PE(克隆R2F2)(AbLab Laboratorio di Istologia e Citologia Patologica Veterinaria,目录号:AB10RS24MW215)
    6. 抗PDGFRα-别藻蓝蛋白(APC,CD140a,clone APA5)(BioLegend,目录号:135907)
    7. 抗LY-6A/E SCA-1-别藻蓝蛋白/Cy7(APC/Cy7,克隆B7)(BD Biosciences,目录号:560654)
    8. 7-氨基放线菌素D(7-AAD)(Life Technologies,目录号:A1310)
  11. 生长培养基(GM)(参见配方)
  12. 洗涤缓冲液(WB)(参见配方)
  13. 胶原酶II溶液(参见配方)
  14. 红细胞裂解缓冲液(见配方)
  15. 排序缓冲区(请参阅配方)
  16. Matrigel解决方案(参见配方)
  17. b-FGF溶液(见配方)


  1. 剪刀和镊子
  2. 细胞培养塑料盘(Corning,Costar )
  3. 六个多井(Corning,Costar ®
  4. 离心机
  5. 50和15 ml塑料管
  6. 18G-10ml注射器
  7. 70μm和40μm细胞过滤帽(BD Biosciences)
  8. Beckam Coulter细胞分选仪MoFlo TM TM/XDP(Beckman Coulter,目录号:ML99030)
  9. 在37℃和5%CO 2/s的细胞培养箱中培养
  10. 显微镜或细胞计数器


  1. 通过递送递增浓度的CO 2并除去后肢肌肉来牺牲小鼠(C57BL/6或MDX)。
  2. 称量肌肉质量。
  3. 将它们放在含有冷PBS的培养皿中。
  4. 去除可见的腱,脂肪组织和血管
  5. 使用弯曲的尖端剪刀将新培养皿(不含PBS)上的肌肉剁碎几分钟,直到组织表现为糊状。
  6. 将组织转移到50ml塑料管中,并添加0.2%胶原酶II在DMEM(无血清)。使用体积为2-4ml胶原酶对于1g肌肉重量:通常每只小鼠允许获得1-1.5g组织。将塑料管放入振荡恒温槽中,在37℃下1小时。
  7. 通过在500rpm离心从消化的材料中分离未消化的。所有离心均在室温下进行。除去上清液(消化的材料),用室温PBS(约50ml PBS/1g肌肉)稀释并通过18G-10ml注射器以产生单细胞悬浮液(通过针约10次传代)。
  8. 加入到未消化的肌肉新鲜胶原酶溶液(使用相同体积的步骤6),并置于振荡恒温浴中,在37℃另外30分钟。然后,重复步骤7.丢弃在第二次离心程序后可能残留的最终未消化的物质
  9. 通过两个消化获得的肌肉浆液通过70μm细胞滤器帽并随后通过40μm细胞滤器帽过滤,使用50ml注射器和50ml塑料管。
  10. 在2,000 rpm离心10分钟,并使用移液管去除上清液。
  11. 通过用1-2ml红细胞裂解缓冲液重悬细胞来消除血红细胞
  12. 通过加入50ml含有2%FBS的PBS稀释红细胞裂解缓冲液,并在室温下以2,000rpm离心10分钟。
  13. 除去上清液后,用移液管加入1ml含有10%FBS的PBS并计数细胞
  14. 离心管2000 rpm离心10分钟,孵育回收的细胞30分钟与抗SM/C2.6-生物素冰上。 在100μl含有2%FBS的PBS中使用1μl抗体用于1×10 6个细胞。
  15. 加入1ml洗涤缓冲液,并以2,000rpm离心5分钟
  16. 除去上清液后,用链霉抗生物素-PE和其它标记抗体[抗CD31-PECy7和抗CD45-PECy7,抗α7-整联蛋白-PE(对于MDX小鼠),抗PDGFRα -APC,抗LY-6A/E SCA-1-APC/Cy7]和与7-氨基放线菌素D一样。如在步骤14中,在100μl中使用1μl抗体用于1×10 6个细胞μl含有2%FBS的PBS。
  17. 加入50ml洗涤缓冲液,并以2,000rpm离心5分钟
  18. 除去上清液后,加入2ml分选缓冲液/小鼠
  19. 为了建立仪器,将约50-100,000个细胞的样品与各单个抗体或用7-氨基放线菌素(单染色补偿)在相同条件的样品中平行分别染色。 50-100,000个细胞的样品也没有与任何抗体孵育(未染色的对照)。 30分钟后,加入1ml洗涤缓冲液,然后离心(2,000rpm,5分钟)。除去上清液,加入200-300μl含2%FBS的PBS。
  20. 对于细胞分选机设置:使用未染色的对照,通过前向散射和侧向散射排除碎片和死细胞并设置基础荧光(图1A)。然后进行7-AAD门控,使用用7- 氨基放线菌素(门1,7AAD阴性细胞和门2),以进一步消除死亡
  21. 使用单独用每种抗体染色的样品补偿荧光,然后在样品中选择以将CD34细胞分选为CD45 -/sup/CD31 -/- >(门3)/smc2.6 (门4)/SCA-1 + 和PDGFRα + (门5)细胞。为了提高纯度,我们优选对WT小鼠使用抗smc2.6,对于mdx 小鼠使用抗α7整联蛋白。
  22. 在分选结束时,在预先用Matrigel包被的多孔平板中以1×10 4个细胞/cm 2的平板FAP细胞在生长培养基中加上b- FGF在潮湿培养箱(37℃和5%CO 2)中培养


图1.细胞分选程序程序。 一个。单细胞门控; B.基于选择7ADD阴性细胞(1,左)和前向散射/侧向散射参数(2,右)选择的活细胞; CD31和CD45阴性细胞门控(3); D.α7整联蛋白门控(4); E.卫星细胞门控(Sca 1 /来自α7-整合素阳性门的PDGFRα阴性细胞); FAP细胞注射(ScA 1 /PDGFRα阳性细胞来自α7-整合素阴性门(5)。


  1. FAP细胞回收范围为总细胞的1至3%。 消化似乎是关键点。


  1. 生长培养基(GM)
  2. 洗涤缓冲液(WB)
    PBS w/o Ca ++ 和Mg ++ ,加热灭活2%FBS。
  3. 胶原酶II溶液
  4. 红细胞裂解缓冲液
    0.8%NH 4 Cl的Tris缓冲液(pH 8)中
  5. 排序缓冲区
    PBS w/o Ca 2+ 2 + 和Mg 2+ 2+ 与5%热灭活的FBS接触。
  6. Matrigel解决方案
    在DMEM中稀释Matrigel 1:100,并在制备板涂料后立即使用
  7. b-FGF溶液
    在培养物中,b-FGF浓度为5ng/ml 除霜后,b-FGF等分试样可保持在4℃,并在一周内使用


我们感谢Suk/Fukada教授(大阪大学,大阪,日本)的SMC/2.6抗体。 这项工作得到了欧洲共同体框架计划FP7/2007-2013(授权协议n°241440),意大利卫生部RC 2013,意大利Ricerca sul Cancro(AIRC IG11362)和Ricerca大学部长 PRIN 2010-2011。


  1. Cordani,N.,Pisa,V.,Pozzi,L.,Sciorati,C.and Clementi,E。(2014)。 一氧化氮通过调节成纤维脂肪前体分化来控制营养不良骨骼肌中的脂肪沉积。 32(4):874-885
  2. Fukada,S.,Higuchi,S.,Segawa,M.,Koda,K.,Yamamoto,Y.,Tsujikawa,K.,Kohama,Y.,Uezumi,A.,Imamura,M.,Miyagoe-Suzuki,Y 。,Takeda,S。和Yamamoto,H。(2004)。 通过新型单克隆抗体从鼠骨骼肌的静止卫星细胞的纯化和细胞表面标记物表征。 Exp Cell Res 296(2):245-255。
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引用:Cordani, N., Pisa, V., Pozzi, L. and Sciorati, C. (2014). Isolation of FAP Cells from Mouse Dystrophic Skeletal Muscle Using Fluorescence Activated Cell Sorting. Bio-protocol 4(22): e1292. DOI: 10.21769/BioProtoc.1292.



Huan Li
Sun Yat-sen University
I want to know the size of FAP cells since the information is important for my experiment. I am looking forward to the answer. Thank you!
5/11/2018 9:43:50 PM Reply
Clara Sciorati
Division of Regenerative Medicine, Ospedale San Raffaele, Italy

In plate FAP cells are around 20-25um.
All my best and cross the finger for your experiment!

5/27/2018 12:25:52 PM

Clara Sciorati
Division of Regenerative Medicine, Ospedale San Raffaele, Italy

what do you mean for size? they are quite similar to satellite cells...

5/27/2018 11:39:16 PM

Huan Li
Sun Yat-sen University

Thank you for your answer! It is helpful.

5/27/2018 11:46:28 PM