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Ki67 Immunofluorescence on Bovine Cell Lines
Ki67 免疫荧光法检测牛细胞系   

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Abstract

This is a rapid protocol to test the effects of drugs treatment on bovine cell replication using Ki67 staining. Ki67 is associated with cell proliferation and is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent from resting cells (G0).

Keywords: Ki67(Ki67), Immunofluorescence(免疫荧光), Bovine lymphocytes(牛淋巴细胞)

Materials and Reagents

  1. Bovine cells (The TBL3 cell line was derived from in vitro infection of the spontaneous bovine-B lymphosarcoma cell line, BL3, with Hissar stock of T. annulata)
  2. RPMI 1640 (Gibco)
  3. Fetal calf serum (heat-inactivated)
  4. Penicillin/streptomycin
  5. Fibronectin (Sigma-Aldrich, catalog number: F1141 )
  6. Formaldehyde (Sigma-Aldrich, catalog number: F8775 )
  7. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
  8. SVF (PAA Laboratories GmbH, catalog number: A15-101 )
  9. BSA (Sigma-Aldrich, catalog number: A2153 )
  10. Mouse monoclonal anti-Ki67 (1:50) (Abcam, catalog number: ab10913-1 )
  11. Cy2 AffinyPure anti-mouse IgG (1:5,000) (Jackson ImmunoResearch Laboratories, catalog number: 715-225-150 )
  12. Tween 20 (Biosolve, catalog number: 2045 2335 )
  13. ProLong Gold Antifade Reagent with Dapi (Life Technologies, Invitrogen™, catalog number: P-36931 )
  14. DAPI

Equipment

  1. Slides (Knittel Glass, catalog number: KN00010025787 )
  2. 37 °C, 5% CO2 cell culture incubator
  3. 24 wells plate
  4. Fluorescent microscope (Leica Microsystems, model: Inverted 6000 )

Procedure

  1. Bovine cells were cultured in RPMI 1640, supplemented with 10% heat-inactivated Fetal calf serum, 4 mM L-Glutamine, 25 mM HEPES, 10 μM β-mercaptoethanol  and 100 μg/ml penicillin/streptomycin in a humidified 5% CO2 atmosphere at 37 °C.
  2. In 24 wells plate, slides were coated with PBS – Fibronectin (1/1,000), 2 h at 37 °C and wash twice with PBS.
  3. Non adherent bovine cells (500,000 cells/well in 24 wells plate) were plated on Fibronectin coated slides 2 h at 37 °C, 5% CO2 and then fixed in 500 μl PBS 3.7% Formaldehyde for 15 min at room temperature.
  4. Slides were rinsed in PBS (300 μl - 3 washes) and permeabilized with PBS 0.2% Triton X-100 for 5 min.
  5. Slides were washed in PBS (300 μl - 3 washes) and then blocked for 30 min at room temperature with 250 μl PBS 1% SVF and 1% BSA to prevent non-specific staining.
  6. The slides were incubated with Mouse monoclonal anti-Ki67 (1:50) in 100 μl PBS 1% SVF and 1% BSA at room temperature for 40 min.
  7. After washing in PBS 0.2% Tween (300 μl - 3 washes), the slides were incubated with Cy2 AffinyPure anti-mouse IgG (1:5,000) in 100 μl PBS 1% SVF and 1% BSA for 30 min at room temperature, in the dark.
  8. Slides were subsequently washed in PBS 0.2% Tween (300 μl - 3 washes), mounted on slides and coverslippedwith ProLong Gold Antifade Reagent with DAPI.
  9. Images of immunofluorescence staining were photographed with a camera attached to a fluorescent microscope and percentage of Ki67 positive cells was calculated.
  10. This staining was repeated for three independent biological replicates (Figure 1).


    Figure 1. Ki67 staining of bovine cells

Acknowledgments

This protocol was adapted form James et al. (2005). The Weitzman laboratory was supported by Fondation de France (FdF #2102), Association pour le Recherche contre le Cancer (ARC Fixe #4975, ARC-Equipement #7990) and Association for International Cancer Research (AICR, #08-0111).

References

  1. James, D., Levine, A. J., Besser, D. and Hemmati-Brivanlou, A. (2005). TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development 132(6): 1273-1282.
  2. Marsolier, J., Pineau, S., Medjkane, S., Perichon, M., Yin, Q., Flemington, E., Weitzman, M. D. and Weitzman, J. B. (2013). OncomiR addiction is generated by a miR-155 feedback loop in Theileria-transformed leukocytes. PLoS Pathog 9(4): e1003222.   

简介

这是一个快速协议,以测试药物治疗对牛细胞复制使用Ki67染色的影响。 Ki67与细胞增殖相关,并且在细胞周期(G1,S,G2和有丝分裂)的所有活性期期间存在,但在静息细胞(G0)中不存在。

关键字:Ki67, 免疫荧光, 牛淋巴细胞

材料和试剂

  1. 牛细胞(TBL3细胞系来源于自发性牛-B淋巴肉瘤细胞系BL3的体外感染,用Hissar股票新戊型肝炎病毒感染)
  2. RPMI 1640(Gibco)
  3. 胎牛血清(热灭活)
  4. 青霉素/链霉素
  5. 纤连蛋白(Sigma-Aldrich,目录号:F1141)
  6. 甲醛(Sigma-Aldrich,目录号:F8775)
  7. Triton X-100(Sigma-Aldrich,目录号:T9284)
  8. SVF(PAA Laboratories GmbH,目录号:A15-101)
  9. BSA(Sigma-Aldrich,目录号:A2153)
  10. 小鼠单克隆抗Ki67(1:50)(Abcam,目录号:ab10913-1)
  11. Cy2 AffinyPure抗小鼠IgG(1:5,000)(Jackson ImmunoResearch Laboratories,目录号:715-225-150)
  12. Tween 20(Biosolve,目录号:2045 2335)
  13. ProLong Gold Antifade Reagent with Dapi(Life Technologies,Invitrogen TM,目录号:P-36931)
  14. DAPI

设备

  1. 幻灯片(Knittel Glass,目录号:KN00010025787)
  2. 37℃,5%CO 2细胞培养箱中培养
  3. 24孔板
  4. 荧光显微镜(Leica Microsystems,型号:Inverted 6000)

程序

  1. 牛细胞在补充有10%热灭活的胎牛血清,4mM L-谷氨酰胺,25mM HEPES,10μMβ-巯基乙醇的RPMI 1640中培养。 和100μg/ml青霉素/链霉素的DMEM培养基中在37℃的湿润的5%CO 2气氛中培养。
  2. 在24孔板中,用PBS-纤连蛋白(1/1000)包被载玻片,在37℃下2小时,并用PBS洗涤两次。
  3. 将非粘附牛细胞(24孔板中的500,000个细胞/孔)在37℃,5%CO 2下接种在纤维连接蛋白包被的载玻片上2小时,然后固定在500μlPBS 3.7%甲醛中15分钟min。
  4. 将载玻片在PBS(300μl-3次洗涤)中漂洗并用PBS 0.2%Triton X-100透化5分钟。
  5. 将载玻片在PBS(300μl-3次洗涤)中洗涤,然后在室温下用250μlPBS 1%SVF和1%BSA封闭30分钟以防止非特异性染色。
  6. 将载玻片与小鼠单克隆抗Ki67(1:50)在100μlPBS 1%SVF和1%BSA中在室温下温育40分钟。
  7. 在PBS 0.2%吐温(300μl-3次洗涤)中洗涤后,将载玻片与在100μlPBS 1%SVF和1%BSA中的Cy2 AffinyPure抗小鼠IgG(1:5,000)在室温下温育30分钟,黑暗。
  8. 随后将载玻片在PBS 0.2%吐温(300μl-3次洗涤)中洗涤,安装在载玻片上,并用带有DAPI的ProLong Gold Antifade试剂盖上盖子。
  9. 用连接到荧光显微镜的照相机拍摄免疫荧光染色的图像,并计算Ki67阳性细胞的百分比。
  10. 对三个独立的生物学重复(图1)重复该染色

    图1.牛细胞的Ki67染色

致谢

该协议改编自James 等人(2005)。 Weitzman实验室由Fondation de France(FdF#2102),癌症协会(ARC Fixe#4975,ARC-Equipement#7990)和国际癌症研究协会(AICR,#08-0111)支持。

参考文献

  1. James,D.,Levine,A.J.,Besser,D。和Hemmati-Brivanlou,A。(2005)。 TGFbeta/activin/nodal信号是维持人胚胎干细胞多能性所必需的。 a> 132(6):1273-1282。
  2. Marsolier,J.,Pineau,S.,Medjkane,S.,Perichon,M.,Yin,Q.,Flemington,E.,Weitzman,M.D.and Weitzman,J.B。(2013)。 OncomiR成瘾由miR-155反馈回路产生,其中 转化的白细胞。 PLoS Pathog 9(4):e1003222。   
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Marsolier, J., Medjkane, S., Perichon, M. and Weitzman, J. B. (2013). Ki67 Immunofluorescence on Bovine Cell Lines. Bio-protocol 3(21): e958. DOI: 10.21769/BioProtoc.958.
  2. Marsolier, J., Pineau, S., Medjkane, S., Perichon, M., Yin, Q., Flemington, E., Weitzman, M. D. and Weitzman, J. B. (2013). OncomiR addiction is generated by a miR-155 feedback loop in Theileria-transformed leukocytes. PLoS Pathog 9(4): e1003222.
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