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Jul 2017

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A Method for Culturing Mouse Whisker Follicles to Study Circadian Rhythms ex vivo
一种离体研究昼夜节律的小鼠触须毛囊培养方法   

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Abstract

Ex vivo tissue-culture experiments are often performed in the field of circadian biology. The major aim of these experiments is to evaluate circadian characteristics such as period length at the tissue-autonomous level by monitoring clock gene expression in real time. This culture method is also used to examine the tissue specificity of circadian entrainment factors. However, an ex vivo culture method for monitoring clock gene expression in hair follicles has yet to be established. In the present study, we developed an experimental method to analogize and evaluate circadian characteristics by performing ex vivo culture of mouse whisker follicles and monitoring clock gene expression in real time.

Keywords: Circadian rhythm (昼夜节律), Clock gene (时钟基因), Hair follicle (毛囊), Ex vivo culture (体外培养), Luciferase (荧光素酶), Mouse (小鼠)

Background

Almost all living organisms exhibit physiological and behavioral circadian rhythms that are driven by the circadian clock (Takahashi, 2017). The circadian clock enables maximum expression of genes at appropriate times of the day, allowing organisms to appropriately adapt to environmental rhythms generated by the Earth’s rotation. The clockwork consists of ubiquitous, cell-autonomous and clock gene-driven negative feedback loops of transcription (Schibler et al., 2015). In mammals, the transcription factors BMAL1 and CLOCK activate the transcription of clock and clock-related genes such as Period (Per) and Cryptochrome (Cry) via E-box elements. PER, together with CRY, a potent transcriptional inhibitor, subsequently function to negatively regulate this complex (Kume et al., 1999).

Ex vivo tissue-culture experiments are often performed in the field of circadian biology (Yamazaki et al., 2000). The major aim of these experiments is to evaluate the circadian characteristics of clock gene expression such as period length at the tissue-autonomous level and to compare these characteristics with those at the whole-body level (Liu et al., 2007). For example, the effect of the dysfunction of a clock gene in question can be investigated by comparing the effects in ex vivo tissue culture and behavior and physiology. This culture method is also used to examine the tissue specificity of circadian entrainment factors (Sato et al., 2014). Specifically, this technique can be used to reveal in which tissue a humoral factor in question modulates circadian phase or amplitude. Additionally, although controversial, some studies suggest that the circadian phase observed in ex vivo cultured tissues can be used to estimate that in vivo (Stokkan et al., 2001).

In the present study, we developed an experimental method to analogize and evaluate circadian characteristics based on ex vivo culture of mouse whisker follicles. Briefly, individual whisker follicles are carefully dissected from mice carrying a luciferase gene whose expression is driven by a circadian promoter, and bioluminescence is measured in real time using a photomultiplier tube. This method can be useful for a wide range of applications as mentioned above.

Materials and Reagents

  1. 100 mm Petri dish (IWAKI, catalog number: SH90-20)
  2. 35 mm culture dish (IWAKI, catalog number: 1000-035)
  3. Eppendorf tube
  4. Inbred mice such as male 5-20-week-old C57/BL6 mice carrying the luciferase gene driven by a clock gene promoter (in our original paper, we used Per2::luc knock-in mice and Bmal1-Eluc transgenic mice, which were gifts from Dr. Joseph Takahashi and Dr. Yoshihiro Nakajima, respectively) (Yoo et al., 2004; Noguchi et al., 2012a)
  5. Silicone (Shin-Etsu, catalog number: KS-64)
  6. 70% ethanol (Shinwa Alcohol Industry, catalog number: 4079210060)
  7. Phosphate-buffered saline (PBS) (Nacalai Tesque, catalog number: 14249-24)
  8. DMEM (Nacalai, catalog number: 08456-94)
  9. Penicillin/streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15070-063)
  10. Dexamethasone (Sigma-Aldrich, catalog number: D4902)
  11. Luciferin (WAKO, catalog number: 126-05116)
  12. Dimethyl sulfoxide
  13. DMEM supplemented with penicillin/streptomycin (see Recipes)
  14. Luciferin-containing medium (see Recipes)
  15. 1,000x DEX stock (see Recipes)

Equipment

  1. Surgical scissors (Hammacher, catalog number: 91-1538)
  2. Claw tweezers (FST, catalog number: 11154-10)
  3. Fine tweezers (FST, catalog number: 18132-12)
  4. Laminar flow cabinet (SANYO, model: MCV-B131F)
  5. CO2 incubator with an infrared sensor that is not affected by humidity inside the chamber (ASTEC, model: SCA-165DRS)
  6. Photomultiplier tube (Hamamatsu, model: LM2400)
  7. Dissecting microscope (Nikon, model: SMZ745T)

Software

  1. Software for Photon Detection Unit C10749(LM2400v21)-JP
  2. Cosinor software

Procedure

Notes:

  1. Although the absolute value of circadian period length in cultured hair follicles differs from those obtained from physiological and behavioral studies, we confirmed that the relative difference in period length among mouse genotypes is similar between clock gene expression in hair follicles and locomotor activity (Yamaguchi et al., 2017). Our ex vivo methods may, therefore, be useful tools for analogizing in vivo circadian characteristics.
  2. Because culture medium composition reportedly affects period length (Lee et al., 2011; Noguchi et al., 2012b), it is important to use identical medium composition and product lot numbers throughout all sets of experiments for comparison of relative differences between animals. For example, we have found that the absence of phenol red results in damping and relatively longer periods.
  3. Dexamethasone (DEX) is a well-known synchronizer for peripheral clocks. Therefore, clock gene expression is often monitored after DEX treatment.

  1. Isolation of mouse whisker follicles
    1. All protocols for animal experiments must be approved by an institutional animal research committee. Animal studies must be performed in compliance with institutional animal care and use guidelines. Figure 1 indicates a clean environment and tools required for following experimental procedures.


      Figure 1. Example of a clean environment and tools required for the experimental procedures

    2. Maintain mice carrying the luciferase gene driven by circadian promoter/enhancer elements on a 12-h light-dark (LD) cycle and allow ad libitum access to food and water.
    3. After euthanasia, vigorously wipe both the left and right mystacial pads with 70% ethanol and remove them from the mice with surgical scissors by making incisions in the recommended order indicated in Figure 2. To avoid damaging hair follicles, insert surgical scissors and cut along the interface between the skin and bone tissue.


      Figure 2. The mystacial pad on a mouse and the incision line

    4. On a clean bench, vigorously wash the pads two times with 70% ethanol and three times with phosphate-buffered saline (PBS) for about 15 s each (Video 1). Minimize carry-over contamination in each step.

      Video 1. Vigorous washing of mystacial pads

    5. Transfer the pads to a 100-mm Petri dish containing 25 ml fresh DMEM supplemented with penicillin and streptomycin at room temperature. 
    6. On a clean bench, carefully dissect individual whisker follicles as described below under a dissecting microscope (Recommended magnification: 10x).
    7. Use claw tweezers to carefully remove the surrounding connective tissue without damaging the hair follicles (Figure 3A and Video 2). Denude the hair follicles as thoroughly as possible (Figure 3B).


      Figure 3. Dissection of individual whisker follicles. A. Image of the reverse side of a removed mystacial pad. Hair follicles are invisible because they are covered by connective tissue. Use claw tweezers to carefully remove the connective tissue of the mystacial pad without damaging the hair follicles. B. Black outlines indicate denuded hair follicles after removal of the connective tissue. Denude the hair follicles as thoroughly as possible.

      Video 2. Removing connective tissue of the mystacial pad (This video was made at Yamaguchi Univ. according to guidelines from the Yamaguchi Univ. on Animal Care and approved by the Animal Research Ethics Board of Yamaguchi University under protocol #298.)

    8. Hold the root of a hair follicle firmly with fine tweezers and pluck it out from the skin, being careful not to damage the hair follicle (Video 3).

      Video 3. Plucking a hair follicle from the skin (This video was made at Yamaguchi Univ. according to guidelines from the Yamaguchi Univ. on Animal Care and approved by the Animal Research Ethics Board of Yamaguchi University under protocol #298.)

  2. Ex vivo culture of mouse whisker follicles to examine circadian characteristics
    1. Transfer isolated whisker follicles to another 100-mm Petri dish containing 25 ml fresh DMEM supplemented with penicillin and streptomycin at room temperature.
    2. (Optional) Classify the hair cycle stage of whisker follicles as anagen or catagen according to the morphology of the hair bulb and relative length of the hair shaft by referring to previous reports (Iida et al., 2007). We previously investigated the effect of the differences in hair stage on circadian period length and found no significant differences in period length among stages.
    3. Place 50-100 μl silicone into a mound somewhat off-center on the bottom of 35-mm culture dishes. Sterilization of silicone is not essential, but we recommend using silicone from an unopened packet designated for culture use. 
    4. Cut the hair shaft, leaving about 10 to 20 mm (Figure 4A).
    5. Transfer the whisker follicles to the 35-mm culture dishes for bioluminescence monitoring. Use one dish per whisker follicle. To avoid floating during bioluminescence monitoring, push the hair shaft into the silicone mound so that the shaft is stuck to the silicone and is fixed on the bottom of the dish (Figure 4B). Position the hair follicle near the center of the dish for efficient bioluminescence detection (Figure 4C).


      Figure 4. Fixing a hair follicle to the bottom of a culture dish. A. Cut the hair shaft, leaving about 10 to 20 mm. B. Push the hair shaft into the silicone mound so that the shaft is stuck to the silicone and is fixed to the bottom of the dish. C. Position the hair follicle near the center of the dish.

    6. Cover immobilized hair follicles with 3 ml fresh DMEM supplemented with penicillin and streptomycin, and pre-culture at 35 °C with 5% CO2 to allow them to recover from surgical damage. There are no specific parameters for confirming whether or not hair follicles are healthy. 
    7. After 1 or 2 days of pre-culture, induce circadian synchronization by adding 100 nM dexamethasone (DEX) without replacing the culture medium and incubating for 2 h. To facilitate mixing of DEX with culture medium, pipette 500 μl of medium from each culture dish, mix this with 3 μl DEX (100 μM stock) by pipetting in an Eppendorf tube and add this mixture to the original culture dish.
    8. During DEX treatment, prepare luciferin-containing medium at a concentration of 0.1 mM.
    9. Aspirate the DEX-containing medium and wash the hair follicles with 3 ml fresh DMEM to remove DEX, and cover the follicles with 3 ml luciferin-containing medium. Bioluminescence is detectable for a period of more than four days if hair follicles are healthy.
    10. Measure bioluminescence in real time using a photomultiplier tube inside a dark box specifically designed to reduce background noise to detect ultra-weak photon emissions (LM2400, Hamamatsu) at 35 °C with 5% CO2 (Figure 5A). To avoid rust formation, the LM2400 is located inside a CO2 culture incubator under low humidity conditions: to prevent culture medium from drying out, instead of using the water tray from the CO2 incubator, use the one inside the LM2400 (Figures 5B, sterile water). Use a CO2 incubator that utilizes an infrared sensor that is not affected by humidity inside the chamber. For measurement, open the top of the LM2400 and simply place culture dishes on the metal tray (Figures 5B and 5C). Culture dishes without hair follicles should provide reads of 5,000-10,000 counts per minute (negative control). Small lung explants (1-2 mm3) are recommended as a positive control because samples can easily be prepared by simply cutting lung tissue with a surgical knife, and the success rate for obtaining clear circadian oscillations of bioluminescence is high (> 80%). The success rate for detecting clear circadian rhythmicity is dependent on a number of factors such as mouse age, sample handling, and tissue type. Therefore, the required number of sample replicates differs among experiments.


      Figure 5. Monitoring bioluminescence in real time. Bioluminescence can be measured in real time using a photomultiplier tube (LM2400) at 35 °C with 5% CO2. A. The location of photomultiplier tubes (PMTs). Arrows in the inset indicate the photon input windows in a magnified view of two PMTs. B. Culture dishes on the metal tray inside the LM2400. C. Partial enlarged image of B.

Data analysis

Data collection is performed using the associated software “Software for Photon Detection Unit C10749(LM2400v21)-JP”. To analyze circadian parameters, baseline changes need to be removed. Raw data sets are therefore detrended, as shown in Figure 6, using Microsoft Excel by subtracting the 24-h running average from the raw data (Figure 6A, raw data; Figure 6B, detrended data). Circadian robustness, circadian phase (angle) and circadian period length are calculated using detrended data and Cosinor software provided by Dr. Refinetti.


Figure 6. Example data. Data sets are detrended by subtracting the 24-h running average from the raw data. A. Raw data (vertical axis: raw counts per minute); B. Detrended data (vertical axis: detrended relative counts per minute).

Recipes

  1. DMEM supplemented with penicillin and streptomycin
    DMEM
    1% penicillin/streptomycin
  2. Luciferin-containing medium (no filtration required)
    DMEM
    1% penicillin/streptomycin
    0.1 mM luciferin (use 10 mM stock dissolved in saline)
  3. 1,000x DEX stock (no filtration required)
    Dimethyl sulfoxide
    100 μM dexamethasone

Acknowledgments

We thank Ritsuko Matsumura, Rie Okamitsu and Junko Sumino for their expert technical assistance. We express our great appreciation to Takashi Matsuzaki (Shimane University) and Roberto Refinetti (Boise State University) for technical support and Cosinor software, respectively. This protocol was originally developed in Yamaguchi et al. (2017). We acknowledge the support of fellowships from the Yamaguchi Gerontology Research Institute, the Akaeda Medical Research Foundation, the SENSHIN Medical Research Foundation, and the Japan Society for the Promotion of Science.

Competing interests

The authors declare no competing financial interests.

Ethics

All protocols for animal experiments were approved by the Animal Research Committee of Yamaguchi University. Animal studies were performed in compliance with the Yamaguchi University Animal Care and Use guidelines.

References

  1. Iida, M., Ihara, S. and Matsuzaki, T. (2007). Hair cycle-dependent changes of alkaline phosphatase activity in the mesenchyme and epithelium in mouse vibrissal follicles. Dev Growth Differ 49(3): 185-195.
  2. Kume, K., Zylka, M. J., Sriram, S., Shearman, L. P., Weaver, D. R., Jin, X., Maywood, E. S., Hastings, M. H. and Reppert, S. M. (1999). mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop. Cell 98(2): 193-205.
  3. Lee, S. K., Achieng, E., Maddox, C., Chen, S. C., Iuvone, P. M. and Fukuhara, C. (2011). Extracellular low pH affects circadian rhythm expression in human primary fibroblasts. Biochem Biophys Res Commun 416(3-4): 337-342.
  4. Liu, A. C., Welsh, D. K., Ko, C. H., Tran, H. G., Zhang, E. E., Priest, A. A., Buhr, E. D., Singer, O., Meeker, K., Verma, I. M., Doyle, F. J., 3rd, Takahashi, J. S. and Kay, S. A. (2007). Intercellular coupling confers robustness against mutations in the SCN circadian clock network. Cell 129(3): 605-616.
  5. Noguchi, T., Ikeda, M., Ohmiya, Y. and Nakajima, Y. (2012a). A dual-color luciferase assay system reveals circadian resetting of cultured fibroblasts by co-cultured adrenal glands. PLoS One 7(5): e37093.
  6. Noguchi, T., Wang, C. W., Pan, H. and Welsh, D. K. (2012b). Fibroblast circadian rhythms of PER2 expression depend on membrane potential and intracellular calcium. Chronobiol Int 29(6): 653-664.
  7. Sato, M., Murakami, M., Node, K., Matsumura, R. and Akashi, M. (2014). The role of the endocrine system in feeding-induced tissue-specific circadian entrainment. Cell Rep 8(2): 393-401.
  8. Schibler, U., Gotic, I., Saini, C., Gos, P., Curie, T., Emmenegger, Y., Sinturel, F., Gosselin, P., Gerber, A., Fleury-Olela, F., Rando, G., Demarque, M. and Franken, P. (2015). Clock-talk: Interactions between central and peripheral circadian oscillators in mammals. Cold Spring Harb Symp Quant Biol 80: 223-232.
  9. Stokkan, K. A., Yamazaki, S., Tei, H., Sakaki, Y. and Menaker, M. (2001). Entrainment of the circadian clock in the liver by feeding. Science 291(5503): 490-493.
  10. Takahashi, J. S. (2017). Transcriptional architecture of the mammalian circadian clock. Nat Rev Genet 18(3): 164-179.
  11. Yamaguchi, A., Matsumura, R., Matsuzaki, T., Nakamura, W., Node, K. and Akashi, M. (2017). A simple method using ex vivo culture of hair follicle tissue to investigate intrinsic circadian characteristics in humans. Sci Rep 7(1): 6824.
  12. Yamazaki, S., Numano, R., Abe, M., Hida, A., Takahashi, R., Ueda, M., Block, G. D., Sakaki, Y., Menaker, M. and Tei, H. (2000). Resetting central and peripheral circadian oscillators in transgenic rats. Science 288(5466): 682-685.
  13. Yoo, S. H., Yamazaki, S., Lowrey, P. L., Shimomura, K., Ko, C. H., Buhr, E. D., Siepka, S. M., Hong, H. K., Oh, W. J., Yoo, O. J., Menaker, M. and Takahashi, J. S. (2004). PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc Natl Acad Sci U S A 101(15): 5339-5346.

简介

离体组织培养实验通常在昼夜节律生物学领域中进行。 这些实验的主要目的是通过实时监测时钟基因表达来评估昼夜节律特征,例如组织自主水平的周期长度。 该培养方法还用于检查昼夜节律夹带因子的组织特异性。 然而,尚未建立用于监测毛囊中的时钟基因表达的离体培养方法。 在本研究中,我们开发了一种实验方法,通过对小鼠须状卵泡进行离体培养并实时监测时钟基因表达来类比和评估昼夜节律特征。

【背景】几乎所有生物都表现出由生物钟驱动的生理和行为昼夜节律(Takahashi,2017)。生物钟能够在一天中的适当时间最大限度地表达基因,使生物体能够适当地适应地球自转产生的环境节律。发条由普遍存在的,细胞自主的和时钟基因驱动的转录负反馈环组成(Schibler et al。,2015)。在哺乳动物中,转录因子BMAL1和CLOCK激活时钟和时钟相关基因的转录,例如 Period ( Per )和 Cryptochrome ( Cry )通过E-box元素。 PER与CRY一起是一种有效的转录抑制剂,随后起到负调节这种复合物的作用(Kume et al。,1999)。

离体组织培养实验通常在昼夜节律生物学领域进行(Yamazaki 等人,,2000)。这些实验的主要目的是评估时钟基因表达的昼夜节律特征,如组织自主水平的周期长度,并将这些特征与全身水平的特征进行比较(Liu et al。,2007)。例如,可以通过比较离体组织培养和行为和生理学中的作用来研究所讨论的时钟基因的功能障碍的影响。该培养方法还用于检查昼夜节律夹带因子的组织特异性(Sato et al。,2014)。具体地,该技术可用于揭示所讨论的体液因子在哪个组织中调节昼夜节律相位或幅度。此外,虽然有争议,但一些研究表明,离体培养组织中观察到的昼夜节律阶段可用于估计体内 (Stokkan 等。,2001)。

在本研究中,我们开发了一种实验方法,用于基于小鼠须状卵泡的离体培养来类比和评估昼夜节律特征。简而言之,从携带荧光素酶基因的小鼠中仔细解剖单个须状卵泡,所述基因的表达由昼夜节律启动子驱动,并且使用光电倍增管实时测量生物发光。如上所述,该方法可用于广泛的应用。

关键字:昼夜节律, 时钟基因, 毛囊, 体外培养, 荧光素酶, 小鼠

材料和试剂

  1. 100毫米培养皿(IWAKI,目录号:SH90-20)
  2. 35毫米培养皿(IWAKI,目录号:1000-035)
  3. Eppendorf管
  4. 近交小鼠,例如携带由时钟基因启动子驱动的荧光素酶基因的雄性5-20周龄C57 / BL6小鼠(在我们的原始论文中,我们使用 Per2 :: luc 敲入小鼠和 Bmal1-Eluc 转基因小鼠,分别是来自Joseph Takahashi博士和Yoshihiro Nakajima博士的礼物)(Yoo et al。,2004 ; Noguchi et al。,2012a)
  5. 有机硅(信越,产品目录号:KS-64)
  6. 70%乙醇(Shinwa Alcohol Industry,目录号:4079210060)
  7. 磷酸盐缓冲盐水(PBS)(Nacalai Tesque,目录号:14249-24)
  8. DMEM(Nacalai,目录号:08456-94)
  9. 青霉素/链霉素(Thermo Fisher Scientific,Gibco TM ,目录号:15070-063)
  10. 地塞米松(西格玛奥德里奇,目录号:D4902)
  11. Luciferin(WAKO,目录号:126-05116)
  12. 二甲基亚砜
  13. DMEM补充青霉素/链霉素(见食谱)
  14. 含荧光素的培养基(见食谱)
  15. 1,000x DEX库存(见食谱)

设备

  1. 手术剪刀(Hammacher,目录号:91-1538)
  2. 爪式镊子(FST,目录号:11154-10)
  3. 精细镊子(FST,目录号:18132-12)
  4. 层流柜(三洋,型号:MCV-B131F)
  5. CO 2 培养箱,红外传感器不受室内湿度的影响(ASTEC,型号:SCA-165DRS)
  6. 光电倍增管(Hamamatsu,型号:LM2400)
  7. 解剖显微镜(尼康,型号:SMZ745T)

软件

  1. 用于光子探测单元C10749(LM2400v21)-JP的软件
  2. Cosinor软件

程序

注意:

  1. 尽管培养的毛囊中昼夜周期长度的绝对值不同于从生理和行为研究中获得的那些,但我们证实了小鼠基因型的周期长度的相对差异在毛囊中的时钟基因表达和运动活性之间是相似的( Yamaguchi et al。 ,2017)。因此,我们的 离体 方法可能是用于类比 体内 昼夜节律特征的有用工具。
  2. 因为据报道培养基成分会影响周期长度(Lee et al。 ,2011; Noguchi et al。 ,2012b),重要的是在所有实验组中使用相同的培养基组成和产品批号,以比较动物之间的相对差异。例如,我们发现没有酚红会导致阻尼和相对较长的时间。
  3. 地塞米松(DEX)是众所周知的外围时钟同步器。因此,经常在DEX处理后监测时钟基因表达。

  1. 分离小鼠须细胞卵泡
    1. 所有动物实验方案必须得到机构动物研究委员会的批准。动物研究必须遵守机构动物护理和使用指南。图1显示了遵循实验程序所需的清洁环境和工具。


      图1.实验程序所需的清洁环境和工具示例

    2. 在12小时光暗(LD)循环中维持携带由昼夜节律启动子/增强子元件驱动的荧光素酶基因的小鼠,并允许随意获取食物和水。
    3. 安乐死后,用70%乙醇剧烈擦拭左右两侧的mystacial垫,用手术剪刀按照图2所示的推荐顺序从小鼠身上取下。为了避免损伤毛囊,插入手术剪刀并切开皮肤和骨组织之间的界面。


      图2.鼠标上的mystacial pad和切口线

    4. 在干净的工作台上,用70%乙醇剧烈洗涤垫两次,用磷酸盐缓冲盐水(PBS)洗涤三次,每次约15秒(视频1)。最大限度地减少每个步骤中的残留污染。
      视频1.大力清洗mystacial垫

    5. 将垫移至含有25ml新鲜DMEM的100mm培养皿中,该培养皿在室温下补充有青霉素和链霉素。 
    6. 在干净的工作台上,在解剖显微镜下仔细解剖下面所述的单个须状卵泡(推荐放大倍数:10倍)。
    7. 使用爪式镊子小心地去除周围的结缔组织,而不会损坏毛囊(图3A和视频2)。尽可能彻底地去除毛囊(图3B)。


      图3.单个晶须卵泡的解剖。 A.移除的mystacial垫反面的图像。毛囊是看不见的,因为它们被结缔组织覆盖。使用爪式镊子小心地去除mystacial垫的结缔组织,而不会损坏毛囊。 B.黑色轮廓表示去除结缔组织后裸露的毛囊。尽可能彻底地去除毛囊。
      视频2.去除mystacial垫的结缔组织(该视频是在Yamaguchi Univ。根据山口大学动物护理指南制定的,并经山口大学动物研究伦理委员会根据方案#298批准。)
    8. 用细镊子牢牢抓住毛囊的根部并将其从皮肤上拔出,注意不要损伤毛囊(视频3)。
      视频3.从皮肤上采集毛囊(该视频是在山口大学根据山口大学动物护理的指导制作的,并由Yamaguchi大学动物研究伦理委员会根据方案#298批准。)

  2. 离体培养小鼠须细胞卵泡以检查昼夜节律特征
    1. 将分离的须状毛囊转移至另一个100-mm培养皿中,该培养皿含有25ml在室温下补充有青霉素和链霉素的新鲜DMEM。
    2. (可选)参考之前的报道,根据毛球的形态和毛干的相对长度,将毛囊毛囊的毛发周期阶段分为毛发生长期或毛发生长期(Iida et al。,2007) 。我们之前研究了毛发阶段差异对昼夜节律长度的影响,发现阶段间的长度没有显着差异。
    3. 将50-100μl硅胶置于35mm培养皿底部偏离中心的丘中。硅胶灭菌不是必需的,但我们建议使用未开封的指定用于培养的包装中的硅胶。 
    4. 剪掉毛干,留下约10至20毫米(图4A)。
    5. 将须状卵泡转移至35-mm培养皿进行生物发光监测。每个胡须卵泡使用一个盘子。为了避免在生物发光监测过程中漂浮,将毛干推入硅胶丘中,使轴粘在硅胶上并固定在培养皿的底部(图4B)。将毛囊定位在培养皿中心附近,以进行有效的生物发光检测(图4C)。


      图4.将毛囊固定在培养皿的底部。 A.剪掉毛干,留下约10至20毫米。 B.将发干推入硅胶墩,使轴粘在硅胶上,并固定在烤盘底部。 C.将毛囊放在培养皿中心附近。

    6. 用3ml新鲜的补充有青霉素和链霉素的DMEM覆盖固定的毛囊,并在35℃下用5%CO 2 进行预培养,以使它们从手术损伤中恢复。没有确定毛囊是否健康的具体参数。 
    7. 在预培养1或2天后,通过添加100nM地塞米松(DEX)诱导昼夜同步,而不更换培养基并孵育2小时。为了促进DEX与培养基的混合,移取来自每个培养皿的500μl培养基,通过在Eppendorf管中移液将其与3μlDEX(100μM储液)混合,并将该混合物加入到原始培养皿中。
    8. 在DEX处理期间,制备浓度为0.1mM的含荧光素的培养基。
    9. 吸出含有DEX的培养基并用3ml新鲜DMEM洗涤毛囊以除去DEX,并用含有3ml荧光素的培养基覆盖毛囊。如果毛囊是健康的,则可检测到生物发光超过四天的时间。
    10. 使用暗盒内的光电倍增管实时测量生物发光,专门设计用于降低背景噪声,以检测35°C时5%CO 2 的超弱光子发射(LM2400,Hamamatsu)(图5A) )。为了避免生锈,LM2400在低湿度条件下位于CO 2 培养箱内:防止培养基变干,而不是使用CO 2中的水盘培养箱,使用LM2400内的一个(图5B,无菌水)。使用CO 2 培养箱,该培养箱使用不受室内湿度影响的红外传感器。测量时,打开LM2400的顶部,只需将培养皿放在金属托盘上(图5B和5C)。没有毛囊的培养皿应提供每分钟5,000-10,000个读数(阴性对照)。小肺外植体(1-2 mm 3 )被推荐作为阳性对照,因为通过简单地用手术刀切割肺组织可以容易地制备样品,并且获得生物发光的明显昼夜节律振荡的成功率很高(> 80%)。检测清晰的昼夜节律性的成功率取决于许多因素,例如小鼠年龄,样品处理和组织类型。因此,所需的样品重复数量在实验中不同。


      图5.实时监测生物发光。生物发光可以使用光电倍增管(LM2400)在35°C和5%CO 2 下实时测量。 A.光电倍增管(PMT)的位置。插图中的箭头以两个PMT的放大视图指示光子输入窗口。 B. LM2400内部金属托盘上的培养皿。 C. B.的局部放大图像

数据分析

使用相关软件“用于光子检测单元的软件C10749(LM2400v21)-JP”执行数据收集。要分析昼夜节律参数,需要删除基线变化。因此,如图6所示,使用Microsoft Excel从原始数据中减去24小时运行平均值(图6A,原始数据;图6B,去趋势数据),原始数据集被去趋势化。使用由Refinetti博士提供的去趋势数据和Cosinor软件计算昼夜节律稳健性,昼夜节律阶段(角度)和昼夜周期长度。


图6.示例数据。通过从原始数据中减去24小时运行平均值来去除数据集。 A.原始数据(纵轴:每分钟原始计数); B.去趋势数据(纵轴:每分钟去趋势的相对计数)。

食谱

  1. DMEM补充青霉素和链霉素
    DMEM
    1%青霉素/链霉素
  2. 含荧光素的培养基(无需过滤)
    DMEM
    1%青霉素/链霉素
    0.1 mM荧光素(使用溶于盐水的10 mM原液)
  3. 1,000x DEX库存(无需过滤)
    二甲基亚砜
    100μM地塞米松

致谢

我们感谢Ritsuko Matsumura,Rie Okamitsu和Junko Sumino的专业技术支持。我们非常感谢Takashi Matsuzaki(岛根大学)和Roberto Refinetti(博伊西州立大学)获得技术支持和Cosinor软件。该协议最初是在Yamaguchi et al。(2017)中开发的。我们感谢山口老年学研究所,Akaeda医学研究基金会,SENSHIN医学研究基金会和日本科学促进会的奖学金。

利益争夺

作者声明没有竞争性的经济利益。

伦理

所有动物实验方案均经山口大学动物研究委员会批准。根据山口大学动物护理和使用指南进行动物研究。

参考

  1. Iida,M.,Ihara,S。和Matsuzaki,T。(2007)。 毛发周期依赖性碱性磷酸酶活性在小鼠触须毛囊间充质和上皮变化。 Dev Growth Differ 49(3):185-195。
  2. Kume,K.,Zylka,M.J.,Sriram,S.,Shearman,L.P.,Weaver,D.R.,Jin,X.,Maywood,E.S.,Hastings,M.H。和Reppert,S.M。(1999)。 mCRY1和mCRY2是生物钟反馈循环的负面部分的重要组成部分。 Cell 98(2):193-205。
  3. Lee,S.K.,Achieng,E.,Maddox,C.,Chen,S.C.,Iuvone,P.M。和Fukuhara,C。(2011)。 细胞外低pH值会影响人原代成纤维细胞的昼夜节律表达。 Biochem Biophys Res Commun 416(3-4):337-342。
  4. Liu,AC,Welsh,DK,Ko,CH,Tran,HG,Zhang,EE,Priest,AA,Buhr,ED,Singer,O.,Meeker,K.,Verma,IM,Doyle,FJ,3rd,Takahashi, JS和Kay,SA(2007)。 细胞间偶联可提供针对SCN生物钟网络突变的稳健性。 Cell 129(3):605-616。
  5. Noguchi,T.,Ikeda,M.,Ohmiya,Y。和Nakajima,Y。(2012a)。 双色荧光素酶检测系统通过共培养的肾上腺显示培养的成纤维细胞的昼夜节律重置。 / a> PLoS One 7(5):e37093。
  6. Noguchi,T.,Wang,C.W.,Pan,H。和Welsh,D.K。(2012b)。 PER2表达的成纤维细胞昼夜节律取决于膜电位和细胞内钙。 Chronobiol Int 29(6):653-664。
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  10. Takahashi,J。S.(2017)。 哺乳动物生物钟的转录结构。 Nat Rev Genet 18(3):164-179。
  11. Yamaguchi,A.,Matsumura,R.,Matsuzaki,T.,Nakamura,W.,Node,K。和Akashi,M。(2017)。 使用离体培养毛囊组织来研究内在的一种简单方法人类的昼夜节律特征。 Sci Rep 7(1):6824。
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引用:Nishida, A., Miyawaki, Y., Node, K. and Akashi, M. (2019). A Method for Culturing Mouse Whisker Follicles to Study Circadian Rhythms ex vivo. Bio-protocol 9(2): e3148. DOI: 10.21769/BioProtoc.3148.
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