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Oct 2017
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Characterization of Mouse Adult Testicular Macrophage Populations by Immunofluorescence Imaging and Flow Cytometry
免疫荧光成像和流式细胞术鉴定小鼠成人睾丸巨噬细胞群体   

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Abstract

Testicular macrophages (tMΦ) are the most abundant immune cells residing in the testis, an immune-privileged organ. TMΦ are known to exhibit different functions, such as protecting spermatozoa from auto-immune attack by producing immunosuppressive cytokines and trophic roles in supporting spermatogenesis and male sex hormone production. They also contribute to fetal testicular development. Recently, we characterized two distinct tMΦ populations based on their morphology, localization, cell surface markers, and gene expression profiling. Here, we focus and describe in detail the phenotypical distinction of these two tMΦ populations by fluorescence-activated cell sorting (FACS) using multicolor panel antibodies combining with high-resolution immunofluorescence (IF) imaging. These two techniques enable to classify two tMΦ populations: interstitial tMΦ and peritubular tMΦ.

Keywords: Macrophage (巨噬细胞), Testis (睾丸), FACS (FACS), IF ( IF), Spermatogenesis (精子发生), Immune-privilege organ (免疫赦免器官)

Background

The testis is the male reproductive organ where spermatogenesis and testosterone production occurs. tMΦ might be considered as “guardians of fertility” by their immuno-suppressive function to assure the immune-privilege status of the testis and their trophic roles in spermatogenesis and male hormone production, but the distinct functions of these macrophage populations are only starting to be elucidated (Mossadegh-Keller and Sieweke, 2018). Tissue-resident macrophages exhibit tissue-specific functions and gene expression patterns depending of their organs of residency (Gentek et al., 2014; Lavin et al., 2014) but also share common core tissue-resident macrophage markers as F4/80, CD11b, CD64, M-CSFR and for some tissues CX3CR1 (Gordon, 2002; Gautier et al., 2012; Yona et al., 2013; Gentek et al., 2014). Recently, two testicular macrophages (tMΦ) populations have been characterized using IF imaging and FACS (DeFalco et al., 2015; Mossadegh-Keller et al., 2017). Interstitial tMΦ can be identified by microscopy by their rounded morphology and localization in the interstitial space of the testis in close contact with testosterone-producing Leydig cells (Smith and Walker, 2014; DeFalco et al., 2015; Mossadegh-Keller et al., 2017). In contrast, the peritubular tMΦ exhibit an elongated morphology and surround the seminiferous tubules housing spermatogonial stem cells (SSC) (DeFalco et al., 2015; Mossadegh-Keller et al., 2017). Confirming previous observations (DeFalco et al., 2015), we observed that both tMΦ were positive for CX3CR1 and F4/80 by IF. Interstitial tMΦ can be distinguished by the strong expression of M-CSFR from peritubular tMΦ that selectively express high levels of MHCII (Figure 2) (Mossadegh-Keller et al., 2017). We further analyzed in depth the interstitial and peritubular tMΦ populations by FACS. We established an extended antibody panel going beyond previous protocols (DeFalco et al., 2015), excluding monocytes and dendritic cells and including CD64 as a key tissue-resident macrophage (Gautier et al., 2012; Mossadegh-Keller et al., 2017). Within the tMΦ CD45+Ly6C-CD11cloF4/80+CD11b+ fraction, we defined interstitial tMΦ as M-CSFR+CD64hiMHCII- cells and peritubular tMΦ as M-CSFRloCD64loMHCII+ cells (Figure 3).

Here, we describe IF and FACS protocols that will be instrumental to define and isolate these two tMΦ populations for further phenotypic and functional characterization.

Materials and Reagents

  1. Pipette tips
  2. Microscope slides (SuperFrost Plus, VWR, catalog number: 631-0108)
  3. Cover glass (VWR, catalog number: 470820)
  4. Hydrophobic pen, Mini PAP Pen (Life Technologies, catalog number: 008877)
  5. Simport Scientific Disposable Base Molds (Fisher Scientific, catalog number: 11670990)
  6. Eppendorf tube 1.5 ml (Sigma-Aldrich, catalog number: Z606340-1000EA)
  7. Sterilin tube (Thermo Fisher Scientific, catalog number: 129A) 
  8. 5 ml Falcon Polystyrene Round-Bottom Tube (Fisher Scientific, Corning, catalog number: 352008)
  9. Filter 50 μm Filcon, Sterile, Syringe-type (Becton, catalog number: 340601)
  10. Wild-type C57BL/6J mice from Janvier labs
  11. CX3CR1GFP/+ mice (Jung et al., 2000)
  12. Ice
  13. DPBS 1x (Gibco, Life Technologies, catalog number: 14190-094)
  14. HBSS 1x (Gibco, Life Technologies, catalog number: 14025-050)
  15. BSA (Sigma-Aldrich, catalog number: A2153)
  16. Fetal Calf Serum (Biosera, catalog number: FB-1001/500)
  17. Saponin (Sigma-Aldrich, catalog number: 47036)
  18. Antigenfix (Diapath, catalog number: P0014)
  19. Optimum Cutting Temperature (O.C.T.) Tissue-Tek (VWR, catalog number: 256008-930)
  20. Isopentane (VWR, catalog number: 24872-323)
  21. Antibodies:
    1. Anti-CD16/32 (clone 2.4G2, BD Biosciences, catalog number: 553142, working dilution 1/200)
    2. Anti-CD45-BV421 (clone 30F11, BD Biosciences, catalog number: 560501, working dilution 1/200)
    3. Anti-F4/80-BV785 (clone BM8, BioLegend, catalog number: 123141, working dilution 1/200)
    4. Anti-CD11b-BV605 (clone M1/70, BD Biosciences, catalog number: 563015, working dilution 1/800)
    5. Anti-CD64-PerCP/Cy5.5 (clone X54-5/7.1, BioLegend, catalog number: 139308, working dilution 1/300)
    6. Anti-CD11c-BV711 (clone N418, BioLegend, catalog number: 117349, working dilution 1/300)
    7. Anti-CD115-PE (clone AFS98, eBiosciences, catalog number: 112-1152-82, working dilution 1/200)
    8. Anti-Ly6C-APC/Cy7 (clone HK1.4, BioLegend, catalog number: 128026, working dilution 1/300)
    9. Anti-I-A/I-E (MHCII)-PE/Cy7 (clone M5/114.15.2, BioLegend, catalog number: 107630, working dilution 1/300)
    10. Anti-F4/80-Alexa Fluor647 (clone BM8, BioLegend, catalog number: 123122, working dilution 1/200)
    11. Anti-MCII-efluor450 (clone M5/114.15.2, eBiosciences, catalog number: 48-5321-82, working dilution 1/500)
    12. Anti-M-CSFR (clone C-20, Santa Cruz, catalog number: sc-692, working dilution 1/800)
    13. Anti-rabbit-Alexa Fluor 594 (Jackson Immunoresearch, catalog number: 711-585-152, working dilution 1/500)
  22. Prolong Gold antifade reagent (Life Technologies, catalog number: P36930)
  23. Zombie Violet fixable live/dead staining (BioLegend, catalog number: BLE423113, working dilution 1/1,000)
  24. 30% sucrose (Sigma-Aldrich, catalog number: S9378) (see Recipes)
  25. Blocking Buffer for IF (see Recipes)
  26. Wash Buffer for IF (see Recipes)
  27. Prolong containing Sytox blue dye (Life Technologies, catalog number: S34857) (see Recipes)
  28. Enzymatic digestion Mix (see Recipes)a. Collagenase II (Worthington Biochemicals/Serlabo Technologies, catalog number: LS004174, 125 U/mg dry weight)
    a. Collagenase II (Worthington Biochemicals/Serlabo Technologies, catalog number: LS004174, 125 U/mg dry weight)
    b. DNase I (Roche, catalog number: 10104159001)
    c. Working enzymatic digestion (500 µl/testis sample)

Equipment

  1. Pipettes
  2. Microscope slide holder/box (Heathrow Scientific, catalog number: 15994G)
  3. Vertical plastic staining rack
  4. Dissection scissors (Harvard Apparatus, catalog number: 72-8422)
  5. Milligramme balance (Mettler Toledo, catalog number: 30029085)
  6. SnapFrost80 (Alphelys)
  7. Cryostat (Leica, model: CM3050S)
  8. Confocal microscope (ZEISS, model: LSM 780)
  9. 40x/1.4 oil differential interference contrast objective (Plan-Apochromat)
  10. FACS LSRII instrument (BD)
  11. Thermomixer comfort (Eppendorf, catalog number: 5382000015)
  12. Rocking agitator (VWR, catalog number: 444-0756)
  13. Centrifuge
  14. Vortexer
  15. 4 °C refrigerator
  16. -80 °C freezer

Software

  1. FlowJo (Version 10.0.8)
  2. ImageJ (Version 1.49s, National Institutes of Health)
  3. Adobe Illustrator CS6 (Version 16.0.3)

Procedure

  1. Preparing tissue testis sections for IF (Figure 1)
    1. Euthanize mice by cervical dislocation.
    2. Excise the whole testis organ and place it directly in a Sterilin tube with 1 ml cold Antigenfix. Incubate on a rocking agitator for 3 h at 4 °C.
    3. Wash the fixed testis in cold PBS 1x at 4 °C for 20 min.
    4. Incubate the testis organ in 5 ml of 30% sucrose solution overnight at 4 °C, until the testis sinks completely to the bottom of the tube.
    5. Transfer the testis into disposable base molds in a coronal plane and place immediately into cold (-80 °C) isopentane inside the SnapFrost80. After 2 min, remove the frozen molds and store at -80 °C.
    6. Section the mouse testis at 20 µm-thick with the cryostat on SuperFrost slides and store at -20 °C.


    Figure 1. Coronal testis plane preparation. The testis is placed into disposable base molds with OCT in a coronal plane.

  2. Immunofluorescence (Figure 2)
    1. Place slides in a microscope slide holder/box, delimit the testis section with a hydrophobic pen and add 100 µl PBS 1x per section for 10 min at room temperature (RT).
    2. Incubate in blocking buffer for 1 h at RT.
    3. Wash 3 times with the wash buffer solution for 5 min at RT by moving the slide rack up and down between each wash.
    4. Place wet tissues into the slide holder/box in order to make a humid chamber for the staining.
    5. Incubate with antibodies (100 µl per section) in PBS/0.05% saponin overnight at 4 °C at the following dilutions:
      1. Anti-F4/80-Alexa Fluor647 dilution 1/200
      2. Anti-MCII-efluor450 dilution 1/500
      3. Anti-M-CSFR dilution 1/800
    6. After three washes using the wash buffer for 5 min each at RT, incubate for 1 h at RT with anti-rabbit-Alexa Fluor 594, dilution 1/500, for anti-MCSFR antibody detection.
    7. Wash slides three times with PBS/0.05% saponin for 5 min each at RT by moving the slide rack up and down several times. Place slides on a vertical plastic staining rack, protected from the light, in order to dry the slides.
    8. Mount slides in Prolong containing Sytox blue dye (see Recipes).


    Figure 2. Phenotypic characterization of interstitial and peritubular macrophages by IF. IF imaging of CX3CR1GFP/+ adult mouse testis is revealing morphology, localization, F4/80, MHCII and M-CSFR staining of interstitial and peritubular testicular macrophages. One example of interstitial macrophages is indicated with a blue arrow showing a cell with round morphology and expressing CX3CR1, F4/80 and M-CSFR. One example of peritubular macrophages is indicated with a red arrow showing a cell with elongated morphology and expressing CX3CR1, F4/80 and MHCII. Scale bars = 40 μm.

  3. Fluorescence-Activated Cell Sorting (FACS) (Figure 3)
    1. Euthanize mice by cervical dislocation.
    2. Collect the whole testis organ and place it directly in a 1.5 ml Eppendorf tube in cold PBS containing 1 mg/ml Collagenase II/0.15 mg/ml DNase I. Mechanically dissociate the testis by mincing with scissors directly inside the tube.
    3. Place the minced tissue in a thermomixer at 37 °C for 40 min by shaking for enzymatic digestion.
    4. Transfer the digested testis through a 50 μm filter placed on top of a 5 ml Falcon tube and filter using 1 ml PBS.
    5. Centrifuge the digested tissue in a refrigerated centrifuge for 5 min at 300 x g at 4 °C.
    6. Resuspend the pellet of cells in 200 µl of cold PBS and incubate with blocking anti-CD16/32 antibody, dilution 1/200, leaving it on ice for 15 min.
    7. Wash in 1 ml cold PBS and centrifuge for 5 min at 300 x g at 4 °C.
    8. Incubate single-cell suspensions in cold PBS and stain for expression of surface antigens for 20 min on ice. The following antibodies are used in the same mix preparation:
      1. Anti-F4/80-BV785 dilution 1/200
      2. Anti-CD11b-BV605 dilution 1/800
      3. Anti-CD64-PerCP/Cy5.5 dilution 1/300 
      4. Anti-CD11c-BV711 dilution 1/300 
      5. Anti-M-CSFR-PE dilution 1/200 
      6. Anti-Ly6C-APC/Cy7 dilution 1/300 
      7. Anti-I-A/I-E (MHCII)-PE/Cy7 dilution 1/300 
      8. Anti-CD45-BV421 dilution 1/200
      9. Zombie Violet fixable live/dead cell dye as a viability marker dilution 1/1,000
    9. Wash in 1 ml cold PBS and centrifuge for 5 min at 300 x g at 4 °C. Take up single cell suspension in 800 µl of PBS and pass the solution through a 50 µm filter. The sample is ready for the analysis on FACS. 


    Figure 3. Phenotypic characterization of interstitial and peritubular macrophages by FACS. Gating strategy describing in CD45+Ly6C-CD11c-F4/80+CD11b+ adult mouse testis fraction, the distinction of interstitial M-CSFR+CD64+MHCII- and peritubular M-CSFRloCD64loMHCII+ population, respectively represented by blue and red gates.

Data analysis

Confocal microscopy acquisitions were performed on a confocal microscope (LSM780; ZEISS) at room temperature, and slides were imaged with a 40x/1.4 oil differential interference contrast objective (Plan-Apochromat). Different lasers were used (405 nm, 488 nm, 56 nm, and 633 nm) to excite the fluorophores (Sytox blue, eFluor 450, Alexa Fluor 488, Alexa Fluor 594, and Alexa Fluor 647). Fluorescence was recorded in individual channels acquired in a sequential mode to avoid cross-talk using a highly sensitive 32-channel gallium arsenide phosphide detector. The pinhole was set to 1 airy unit. Image processing was done with ImageJ (National Institutes of Health). Only a median filter was applied to the images to remove salt and pepper noise.
  FACS data were acquired on an LSR II instrument (BD) using violet laser 405 nm, blue laser 488 nm, green laser 561 nm, and red laser 633 nm. FACS data were analyzed using FlowJo software (V10.0.8). The FACS gating strategy to distinguish Interstitial CD64hiMHCII- tMΦ and peritubular CD64loMHCII+ tMΦ of adult mouse testis and a representative FACS profile is shown in Figure 2. This can be used to quantify the proportion of these two tMΦ populations and the details regarding the analysis can be found in the original article (Mossadegh-Keller et al., 2017; e.g., Figures 2A and 2B).

Recipes

  1. 30% sucrose
    30 g sucrose
    Bring the volume to 100 ml with PBS
    Store at 4 °C
  2. Blocking Buffer for IF
    2 g BSA
    1 ml FCS
    0.1 g saponin
    Bring the volume to 100 ml with PBS
    Store at 4 °C
  3. Wash Buffer for IF
    0.05 g saponin
    Bring the volume to 100 ml with PBS
    Store at 4 °C
  4. Prolong containing Sytox blue dye
    1 ml of Prolong without DAPI
    Add 3 µl of Sytox blue dye
    Vortex 3 min
    Add 1 ml Prolong without DAPI
    Store at -20 °C
  5. Enzymatic digestion Mix
    1. Collagenase 50 mg/ml stock solution
      Resuspend 1 g of collagenase in 20 ml of HBSS
      Make aliquots of 1 ml
      Store at -20 °C
    2. DNase I 10 mg/ml stock solution
      Resuspend 100 mg of DNase in 10 ml of HBSS
      Make aliquots of 500 µl
      Store at -20 °C
    3. Working enzymatic digestion (500 µl/testis sample)
      10 µl of collagenase stock solution
      7.5 µl of DNase I stock solution, bring the volume to 500 µl with PBS 

Acknowledgments

We thanks the Centre d’Immunologie de Marseille-Luminy (CIML) flow cytometry and mouse house facilities for support. We acknowledge the PICSL imaging facility of the CIML (ImagImm), member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04).
  This work was supported by institutional grants from Institut National de la. Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, and Aix-Marseille University to the CIML and grants to M.H. Sieweke from the Agence Nationale de la Recherche (ANR-11-BSV3-0026), Fondation pour la Recherche Médicale (DEQ. 20110421320) a INSERM-Helmholtz cooperation grant and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement number 695093 MacAge). M.H. Sieweke has been supported as a BIH-Einstein visiting fellow at the MDC and an Alexander von Humboldt Professor at CRTD/TU Dresden.

Competing interests

The authors declare no competing financial interests.

Ethics

In vivo procedures were performed under specific pathogen-free conditions following protocols approved by the Ethics Committee of Marseille in accordance with institutional, national, and European regulations (approval nos. APAFIS 3292-2015 1221 09359224 and APAFIS 10545-2017 0710 08253541).

References

  1. DeFalco, T., Potter, S. J., Williams, A. V., Waller, B., Kan, M. J. and Capel, B. (2015). Macrophages contribute to the spermatogonial niche in the adult testis. Cell Rep 12(7): 1107-1119.
  2. Gautier, E. L., Shay, T., Miller, J., Greter, M., Jakubzick, C., Ivanov, S., Helft, J., Chow, A., Elpek, K. G., Gordonov, S., Mazloom, A. R., Ma'ayan, A., Chua, W. J., Hansen, T. H., Turley, S. J., Merad, M., Randolph, G. J. and Immunological Genome, C. (2012). Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages. Nat Immunol 13(11): 1118-1128.
  3. Gentek, R., Molawi, K. and Sieweke, M. H. (2014). Tissue macrophage identity and self-renewal. Immunol Rev 262(1): 56-73.
  4. Gordon, S. (2002). Pattern recognition receptors: Doubling up for the innate immune response. Cell 111(7): 927-930.
  5. Jung, S., Aliberti, J., Graemmel, P., Sunshine, M. J., Kreutzberg, G. W., Sher, A. and Littman, D. R. (2000). Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol Cell Biol 20(11): 4106-4114.
  6. Lavin, Y., Winter, D., Blecher-Gonen, R., David, E., Keren-Shaul, H., Merad, M., Jung, S. and Amit, I. (2014). Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment. Cell 159(6): 1312-1326.
  7. Mossadegh-Keller, N. and Sieweke, M. H. (2018). Testicular macrophages: Guardians of fertility. Cell Immunol 330: 120-125.
  8. Mossadegh-Keller, N., Gentek, R., Gimenez, G., Bigot, S., Mailfert, S. and Sieweke, M. H. (2017). Developmental origin and maintenance of distinct testicular macrophage populations. J Exp Med 214(10): 2829-2841.
  9. Smith, L. B. and Walker, W. H. (2014). The regulation of spermatogenesis by androgens. Semin Cell Dev Biol 30: 2-13.
  10. Yona, S., Kim, K. W., Wolf, Y., Mildner, A., Varol, D., Breker, M., Strauss-Ayali, D., Viukov, S., Guilliams, M., Misharin, A., Hume, D. A., Perlman, H., Malissen, B., Zelzer, E. and Jung, S. (2013). Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity 38(1): 79-91.

简介

睾丸巨噬细胞(tMΦ)是存在于睾丸中的最丰富的免疫细胞,睾丸是免疫特权器官。 已知TMΦ表现出不同的功能,例如通过产生免疫抑制细胞因子来保护精子免受自身免疫攻击,并且在支持精子发生和雄性激素产生中具有营养作用。 它们还有助于胎儿睾丸发育。 最近,我们基于它们的形态学,定位,细胞表面标志物和基因表达谱分析了两种不同的tMΦ群体。 在这里,我们通过使用多色板抗体结合高分辨率免疫荧光(IF)成像的荧光激活细胞分选(FACS)来集中和详细描述这两个tMΦ群体的表型区别。 这两种技术能够对两个tMΦ群体进行分类:间质tMΦ和管周tMΦ。
【背景】睾丸是雄性生殖器官,其中发生精子发生和睾酮生成。通过免疫抑制功能可以将tMΦ视为“生育的守护者”,以确保睾丸的免疫特权状态及其在精子发生和雄性激素生成中的营养作用,但这些巨噬细胞群体的独特功能才刚刚开始。阐明(Mossadegh-Keller和Sieweke,2018)。组织驻留巨噬细胞表现出组织特异性功能和基因表达模式,取决于其驻留器官(Gentek et al。,2014; Lavin et al。,2014),但也共有核心组织驻留巨噬细胞标记物为F4 / 80,CD11b,CD64,M-CSFR和某些组织CX3CR1(Gordon,2002; Gautier et al。,2012; Yona et al 。,2013; Gentek et al。,2014)。最近,已经使用IF成像和FACS表征了两个睾丸巨噬细胞(tMΦ)群体(DeFalco 等人,2015; Mossadegh-Keller 等人,2017)。间质性tMΦ可以通过显微镜检查通过它们的圆形形态和睾丸间质空间中的定位与睾酮生成的Leydig细胞紧密接触来鉴定(Smith和Walker,2014; DeFalco 等人,2015; Mossadegh-Keller et al。,2017)。相反,管周tMΦ表现出细长的形态,并围绕着精原细胞干细胞(SSC)的生精小管(DeFalco et al。,2015; Mossadegh-Keller et al。 ,2017)。确认先前的观察结果(DeFalco et al。,2015),我们观察到tMΦ对于CX3CR1均为阳性,而IF为F4 / 80。间质tMΦ可以通过选择性表达高水平MHCII的管周tMΦ的M-CSFR的强表达来区分(图2)(Mossadegh-Keller 等人,2017)。我们通过FACS进一步深入分析了间质和管周tMΦ群体。我们建立了一个超越先前方案的扩展抗体组(DeFalco et al。,2015),不包括单核细胞和树突细胞,并且包括CD64作为关键的组织驻留巨噬细胞(Gautier 等。,2012; Mossadegh-Keller et al。,2017)。在tMΦCD45 + Ly6C - CD11c lo F4 / 80 + CD11b + 我们将间质tMΦ定义为M-CSFR + CD64 hi MHCII - 细胞和管周tMΦ为M-CSFR lo CD64 lo MHCII + 细胞(图3)。

在这里,我们描述了IF和FACS协议,这些协议将有助于定义和分离这两个tMΦ群体,以进一步表型和功能表征。

关键字:巨噬细胞, 睾丸, FACS, IF, 精子发生, 免疫赦免器官

材料和试剂

  1. 移液器吸头
  2. 显微镜载玻片(SuperFrost Plus,VWR,目录号:631-0108)
  3. 盖玻片(VWR,目录号:470820)
  4. 疏水笔,迷你PAP笔(Life Technologies,目录号:008877)
  5. Simport Scientific一次性基础模具(Fisher Scientific,目录号:11670990)
  6. Eppendorf管1.5 ml(Sigma-Aldrich,目录号:Z606340-1000EA)
  7. Sterilin管(赛默飞世尔科技,目录号:129A) 
  8. 5毫升Falcon聚苯乙烯圆底管(Fisher Scientific,Corning,目录号:352008)
  9. 过滤器50μmFilcon,无菌,注射器型(Becton,目录号:340601)
  10. 来自Janvier实验室的野生型C57BL / 6J小鼠
  11. CX3CR1GFP / +小鼠(Jung et al。,2000)
  12. DPBS 1x(Gibco,Life Technologies,目录号:14190-094)
  13. HBSS 1x(Gibco,Life Technologies,目录号:14025-050)
  14. BSA(Sigma-Aldrich,目录号:A2153)
  15. 胎牛血清(Biosera,目录号:FB-1001/500)
  16. 皂甙(西格玛奥德里奇,目录号:47036)
  17. Antigenfix(Diapath,目录号:P0014)
  18. 最佳切割温度(O.C.T.)Tissue-Tek(VWR,目录号:256008-930)
  19. 异戊烷(VWR,目录号:24872-323)
  20. 抗体:
    1. 抗CD16 / 32(克隆2.4G2,BD Biosciences,目录号:553142,工作稀释度1/200)
    2. 抗CD45-BV421(克隆30F11,BD Biosciences,目录号:560501,工作稀释度1/200)
    3. 抗F4 / 80-BV785(克隆BM8,BioLegend,目录号:123141,工作稀释度1/200)
    4. 抗CD11b-BV605(克隆M1 / 70,BD Biosciences,目录号:563015,工作稀释度1/800)
    5. 抗CD64-PerCP / Cy5.5(克隆X54-5 / 7.1,BioLegend,目录号:139308,工作稀释度1/300)
    6. 抗CD11c-BV711(克隆N418,BioLegend,目录号:117349,工作稀释度1/300)
    7. 抗CD115-PE(克隆AFS98,eBiosciences,目录号:112-1152-82,工作稀释度1/200)
    8. 抗Ly6C-APC / Cy7(克隆HK1.4,BioLegend,目录号:128026,工作稀释度1/300)
    9. 抗I-A / I-E(MHCII)-PE / Cy7(克隆M5 / 114.15.2,BioLegend,目录号:107630,工作稀释度1/300)
    10. Anti-F4 / 80-Alexa Fluor647(克隆BM8,BioLegend,目录号:123122,工作稀释度1/200)
    11. 抗MCII-efluor450(克隆M5 / 114.15.2,eBiosciences,目录号:48-5321-82,工作稀释度1/500)
    12. 抗M-CSFR(克隆C-20,Santa Cruz,目录号:sc-692,工作稀释度1/800)
    13. Anti-rabbit-Alexa Fluor 594(Jackson Immunoresearch,目录号:711-585-152,工作稀释度1/500)
  21. Prolong Gold抗褪色试剂(Life Technologies,目录号:P36930)
  22. Zombie Violet可固定活/死染色(BioLegend,目录号:BLE423113,工作稀释度1/1000)
  23. 30%蔗糖(Sigma-Aldrich,目录号:S9378)(参见食谱)
  24. 用于IF的阻塞缓冲区(参见食谱)
  25. 用于IF的洗涤缓冲液(参见食谱)
  26. 延长含Sytox蓝染料(Life Technologies,目录号:S34857)(见食谱)
  27. 酶消化混合物(见食谱)a。胶原酶II(Worthington Biochemicals / Serlabo Technologies,目录号:LS004174,125 U / mg干重)
    一个。胶原酶II(Worthington Biochemicals / Serlabo Technologies,目录号:LS004174,125 U / mg干重)
    湾DNase I(罗氏,目录号:10104159001)
    C。工作酶消化(500μl/睾丸样品)

设备

  1. 移液器
  2. 显微镜载玻片架/盒(Heathrow Scientific,目录号:15994G)
  3. 立式塑料染色架
  4. 解剖剪刀(Harvard Apparatus,目录号:72-8422)
  5. Milligramme balance(Mettler Toledo,目录号:30029085)
  6. SnapFrost80(Alphelys)
  7. 低温恒温器(徕卡,型号:CM3050S)
  8. 共聚焦显微镜(蔡司,型号:LSM 780)
  9. 40x / 1.4油分干涉对比物镜(Plan-Apochromat)
  10. FACS LSRII仪器(BD)
  11. Thermomixer comfort(Eppendorf,目录号:5382000015)
  12. 摇摆搅拌器(VWR,目录号:444-0756)
  13. 离心分离机
  14. 涡流混合器
  15. 4°C冰箱
  16. -80°C冰箱

软件

  1. FlowJo(版本10.0.8)
  2. ImageJ(版本1.49s,国立卫生研究院)
  3. Adobe Illustrator CS6(版本16.0.3)

程序

  1. 为组织睾丸切片准备IF(图1)
    1. 通过颈椎脱臼安乐死小鼠。
    2. 切除整个睾丸器官并将其直接置于含有1 ml冷Antigenfix的Sterilin管中。在4°C下在摇动搅拌器上孵育3小时。
    3. 在冷PBS中将固定的睾丸在4℃下洗涤20分钟。
    4. 将睾丸器官在5ml 30%蔗糖溶液中于4℃孵育过夜,直至睾丸完全沉入管底部。
    5. 将睾丸转移到冠状平面中的一次性基础模具中,并立即放入SnapFrost80内的冷(-80°C)异戊烷中。 2分钟后,取出冷冻模具并储存在-80°C。
    6. 在SuperFrost载玻片上用低温恒温器切割20微米厚的小鼠睾丸,并储存在-20°C。


    图1.冠状面睾丸平面准备。将睾丸放入一次性基础模具中,在冠状面上进行OCT。图1.冠状面睾丸平面准备。将睾丸放入一次性基础模具中,在冠状面上进行OCT。

  2. 免疫荧光(图2)
    1. 将载玻片放入显微镜载玻片架/盒中,用疏水笔划分睾丸切片,每室加入100μlPBS1x,室温(RT)10分钟。
    2. 在室温下在封闭缓冲液中孵育1小时。
    3. 通过在每次洗涤之间上下移动载玻片架,在室温下用洗涤缓冲溶液洗涤3次,持续5分钟。
    4. 将湿纸巾放入载玻片架/盒中,以便形成潮湿的染色室。
    5. 与抗体(每节100μl)在PBS / 0.05%皂苷中孵育过夜,在4°C下进行以下稀释:
      1. 抗F4 / 80-Alexa Fluor647稀释度1/200
      2. 抗MCII-efluor450稀释1/500
      3. 抗M-CSFR稀释1/800
    6. 在室温下使用洗涤缓冲液洗涤3次,每次5分钟后,在室温下用抗兔-Alexa Fluor 594(稀释度1/500)孵育1小时,用于抗MCSFR抗体检测。
    7. 通过上下移动载玻片架几次,在室温下用PBS / 0.05%皂苷洗涤载玻片三次,每次5分钟。将载玻片放在垂直塑料染色架上,避光,以便干燥载玻片。
    8. 在Prolong中装载含有Sytox蓝色染料的载玻片(参见食谱)。


    图2.通过IF对间质和肾小管周围巨噬细胞的表型特征。 CX3CR1GFP / +成年小鼠睾丸的IF成像显示间质和管周睾丸的形态,定位,F4 / 80,MHCII和M-CSFR染色巨噬细胞。间质巨噬细胞的一个实例用蓝色箭头表示,显示具有圆形形态并表达CX3CR1,F4 / 80和M-CSFR的细胞。用红色箭头表示管周巨噬细胞的一个实例,显示具有细长形态并表达CX3CR1,F4 / 80和MHCII的细胞。比例尺=40μm。

  3. 荧光激活细胞分选(FACS)(图3)
    1. 通过颈椎脱臼安乐死小鼠。
    2. 收集整个睾丸器官并将其直接置于含有1mg / ml胶原酶II /0.15mg / ml DNase I的冷PBS中的1.5ml Eppendorf管中。通过用剪刀直接在管内切碎来机械解离睾丸。
    3. 通过摇动进行酶消化,将切碎的组织置于37℃的恒温混匀器中40分钟。
    4. 将消化的睾丸转移至置于5ml Falcon管顶部的50μm过滤器,并使用1ml PBS过滤。
    5. 将消化的组织在冷冻离心机中在4℃下以300 x g离心5分钟。
    6. 将细胞沉淀重悬于200μl冷PBS中,并与阻断的抗CD16 / 32抗体一起孵育,稀释1/200,将其在冰上保持15分钟。
    7. 在1ml冷PBS中洗涤,并在4℃下以300μlg/ g离心5分钟。
    8. 将单细胞悬浮液在冷PBS中孵育并染色以在冰上表达表面抗原20分钟。以下抗体用于相同的混合制剂:
      1. 抗F4 / 80-BV785稀释1/200
      2. 抗CD11b-BV605稀释1/800
      3. 抗CD64-PerCP / Cy5.5稀释1/300 
      4. 抗CD11c-BV711(克隆N418,BioLegend,目录号:117349,工作稀释度1/300)
      5. 抗CD115-PE(克隆AFS98,eBiosciences,目录号:112-1152-82,工作稀释度1/200)
      6. 抗Ly6C-APC / Cy7(克隆HK1.4,BioLegend,目录号:128026,工作稀释度1/300)
      7. 抗I-A / I-E(MHCII)-PE / Cy7(克隆M5 / 114.15.2,BioLegend,目录号:107630,工作稀释度1/300)
      8. Anti-F4 / 80-Alexa Fluor647(克隆BM8,BioLegend,目录号:123122,工作稀释度1/200)
      9. 抗MCII-efluor450(克隆M5 / 114.15.2,eBiosciences,目录号:48-5321-82,工作稀释度1/500)
      10. 抗M-CSFR(克隆C-20,Santa Cruz,目录号:sc-692,工作稀释度1/800)
      11. Anti-Rabbit-Alexa Fluor 594(Jackson Immunoresearch,目录号:711-585-152,工作稀释1/500)
      12. Prolong Gold抗褪色试剂(Life Technologies,目录号:P36930)
      13. Zombie Violet可固定活/死染色(BioLegend,目录号:BLE423113,工作稀释度1/1000)
      14. 30%蔗糖(Sigma-Aldrich,目录号:S9378)(参见食谱)
      15. 用于IF的阻塞缓冲区(参见食谱)
      16. 用于IF的洗涤缓冲液(参见食谱)
      17. 延长含Sytox蓝染料(Life Technologies,目录号:S34857)(见食谱)
      18. 酶消化混合物(见食谱)a。胶原酶II(Worthington Biochemicals / Serlabo Technologies,目录号:LS004174,125 U / mg干重)
        一。胶原酶II(Worthington Biochemicals / Serlabo Technologies,目录号:LS004174,125 U / mg干重)
        湾DNase I(罗氏,目录号:10104159001)
        温度。工作酶消化(500μl/睾丸样品)
      19. 设备

        1. 移液器
        2. 显微镜载玻片架/盒(Heathrow Scientific,目录号:15994G)
        3. 立式塑料染色架
        4. 解剖剪刀(Harvard Apparatus,目录号:72-8422)
        5. Milligramme balance(Mettler Toledo,目录号:30029085)
        6. SnapFrost80(Alphelys)
        7. 低温恒温器(徕卡,型号:CM3050S)
        8. 共聚焦显微镜(蔡司,型号:LSM 780)
        9. 40x / 1.4油分干涉对比物镜(Plan-Apochromat)
        10. FACS LSRII仪器(BD)
        11. Thermomixer comfort(Eppendorf,目录号:5382000015)
        12. 摇摆搅拌器(VWR,目录号:444-0756)
        13. 离心分离机
        14. 涡流混合器
        15. 4°C冰箱
        16. -80°C冰箱

        软件

        1. FlowJo(版本10.0.8)
        2. ImageJ(版本1.49s,国立卫生研究院)
        3. Adobe Illustrator CS6(版本16.0.3)

        程序

        1. 为组织睾丸切片准备IF(图1)
          1. 通过颈椎脱臼安乐死小鼠。
          2. 切除整个睾丸器官并将其直接置于含有1 ml冷Antigenfix的Sterilin管中。在4°C下在摇动搅拌器上孵育3小时。
          3. 在冷PBS中将固定的睾丸在4℃下洗涤20分钟。
          4. 将睾丸器官在5ml 30%蔗糖溶液中于4℃孵育过夜,直至睾丸完全沉入管底部。
          5. 将睾丸转移到冠状平面中的一次性基础模具中,并立即放入SnapFrost80内的冷(-80°C)异戊烷中。 2分钟后,取出冷冻模具并储存在-80°C。
          6. 在SuperFrost载玻片上用低温恒温器切割20微米厚的小鼠睾丸,并储存在-20°C。


          图1.冠状面睾丸平面准备。将睾丸放入一次性基础模具中,在冠状面上进行OCT。图1.冠状面睾丸平面准备。将睾丸放入一次性基础模具中,在冠状面上进行OCT。

        2. 免疫荧光(图2)
          1. 将载玻片放入显微镜载玻片架/盒中,用疏水笔划分睾丸切片,每室加入100μlPBS1x,室温(RT)10分钟。
          2. 在室温下在封闭缓冲液中孵育1小时。
          3. 通过在每次洗涤之间上下移动载玻片架,在室温下用洗涤缓冲溶液洗涤3次,持续5分钟。
          4. 将湿纸巾放入载玻片架/盒中,以便形成潮湿的染色室。
          5. 与抗体(每节100μl)在PBS / 0.05%皂苷中孵育过夜,在4°C下进行以下稀释:
            1. 抗F4 / 80-Alexa Fluor647稀释度1/200
            2. 抗MCII-efluor450稀释1/500
            3. 抗M-CSFR稀释1/800
          6. 在室温下使用洗涤缓冲液洗涤3次,每次5分钟后,在室温下用抗兔-Alexa Fluor 594(稀释度1/500)孵育1小时,用于抗MCSFR抗体检测。
          7. 通过上下移动载玻片架几次,在室温下用PBS / 0.05%皂苷洗涤载玻片三次,每次5分钟。将载玻片放在垂直塑料染色架上,避光,以便干燥载玻片。
          8. 在Prolong中装载含有Sytox蓝色染料的载玻片(参见食谱)。


          图2.通过IF对间质和肾小管周围巨噬细胞的表型特征。 CX3CR1GFP / +成年小鼠睾丸的IF成像显示间质和管周睾丸的形态,定位,F4 / 80,MHCII和M-CSFR染色巨噬细胞。间质巨噬细胞的一个实例用蓝色箭头表示,显示具有圆形形态并表达CX3CR1,F4 / 80和M-CSFR的细胞。用红色箭头表示管周巨噬细胞的一个实例,显示具有细长形态并表达CX3CR1,F4 / 80和MHCII的细胞。比例尺=40μm。

        3. 荧光激活细胞分选(FACS)(图3)
          1. 通过颈椎脱臼安乐死小鼠。
          2. 收集整个睾丸器官并将其直接置于含有1mg / ml胶原酶II /0.15mg / ml DNase I的冷PBS中的1.5ml Eppendorf管中。通过用剪刀直接在管内切碎来机械解离睾丸。
          3. 通过摇动进行酶消化,将切碎的组织置于37℃的恒温混匀器中40分钟。
          4. 将消化的睾丸转移至置于5ml Falcon管顶部的50μm过滤器,并使用1ml PBS过滤。
          5. 将消化的组织在冷冻离心机中在4℃下以300 x g离心5分钟。
          6. 将细胞沉淀重悬于200μl冷PBS中,并与阻断的抗CD16 / 32抗体一起孵育,稀释1/200,将其在冰上保持15分钟。
          7. 在1ml冷PBS中洗涤,并在4℃下以300μlg/ g离心5分钟。
          8. 将单细胞悬浮液在冷PBS中孵育并染色以在冰上表达表面抗原20分钟。以下抗体用于相同的混合制剂:
            1. 抗F4 / 80-BV785稀释1/200
            2. 抗CD11b-BV605稀释1/800
            3. 抗CD64-PerCP / Cy5.5稀释1/300 
            4. 抗CD11c-BV711稀释1/300 
            5. 抗M-CSFR-PE稀释度1/200 
            6. 抗Ly6C-APC / Cy7稀释1/300 
            7. 抗I-A / I-E(MHCII)-PE / Cy7稀释1/300 
            8. 抗CD45-BV421稀释度1/200
            9. Zombie Violet可固定活/死细胞染料作为活力标记稀释度1 / 1,000
          9. 在1ml冷PBS中洗涤,并在4℃下以300μlg/ g离心5分钟。在800μlPBS中吸收单细胞悬浮液,并将溶液通过50μm过滤器。样品已准备好进行FACS分析。 


          图3.通过FACS对间质和管周巨噬细胞的表型特征。在CD45 + Ly6C - CD11c - F4 / 80 + CD11b + 成年小鼠睾丸分数,间质M-CSFR + CD64 + 的区别MHCII - 和管周M-CSFR lo CD64 lo MHCII + 群体,分别由蓝色和红色门表示。

        数据分析

        共聚焦显微镜采集在室温下在共焦显微镜(LSM780; ZEISS)上进行,并且用40x / 1.4油差分干涉对比物镜(Plan-Apochromat)对载玻片成像。使用不同的激光(405nm,488nm,56nm和633nm)来激发荧光团(Sytox blue,eFluor 450,Alexa Fluor 488,Alexa Fluor 594和Alexa Fluor 647)。在顺序模式下获得的各个通道中记录荧光,以避免使用高灵敏度的32通道砷化镓磷化物检测器进行串扰。针孔设置为1个通风单位。使用ImageJ(National Institutes of Health)进行图像处理。仅对图像应用中值滤波器以去除盐和胡椒噪声。
         使用紫外激光405nm,蓝色激光488nm,绿色激光561nm和红色激光633nm在LSR II仪器(BD)上获得FACS数据。使用FlowJo软件(V10.0.8)分析FACS数据。 FACS门控策略,以区分成年小鼠的间质CD64 hi MHCII - tMΦ和肾小管周围CD64 lo MHCII + tMΦ睾丸和代表性FACS图谱如图2所示。这可用于量化这两个tMΦ群体的比例,有关分析的详细信息可以在原始文章中找到(Mossadegh-Keller et al。,2017; 例如。,图2A和2B)。

        食谱

        1. 30%蔗糖
          30克蔗糖
          用PBS将体积调至100毫升
          储存在4°C
        2. 用于阻止缓冲区
          2克BSA
          1毫升FCS
          0.1克皂苷
          用PBS将体积调至100毫升
          储存在4°C
        3. 用于IF的洗涤缓冲液
          0.05克皂苷
          用PBS将体积调至100毫升
          储存在4°C
        4. 延长含有Sytox蓝色染料
          1毫升没有DAPI的延长
          加入3μlStoxx蓝色染料
          旋涡3分钟
          加入1毫升不含DAPI的延长
          储存在-20°C
        5. 酶消化混合
          1. 胶原酶50 mg / ml储备液
            将1克胶原酶重悬于20毫升HBSS中 制作1毫升的等分试样
            储存在-20°C
          2. DNase I 10 mg / ml储备液
            在100毫升HBSS中重悬100毫克DNA酶
            制作500μl的等分试样
            储存在-20°C
          3. 工作酶消化(500μl/睾丸样品)
            10μl胶原酶原液
            7.5μlDNaseI储备液,用PBS将体积调至500μl。

        致谢

        我们感谢Center d'Immunologie de Marseille-Luminy(CIML)流式细胞仪和鼠标室设施的支持。我们感谢CIML(ImagImm)的PICSL成像设施,该设施是法国国家研究机构(ANR-10-INBS-04)支持的国家基础设施France-BioImaging的成员。
         这项工作得到了Institut National de la的机构拨款的支持。 Santéetde laRechercheMédicale,国家科学研究中心和Aix-Marseille大学到CIML并授予M.H. Sieweke来自Agence Nationale de la Recherche(ANR-11-BSV3-0026),Fondation pour laRechercheMédicale(DEQ.20110421320),INSERM-Helmholtz合作拨款和欧洲研究理事会(ERC),隶属于欧盟的Horizon 2020研究和创新计划(拨款协议编号695093 MacAge)。 M.H。 Sieweke作为MDC的BIH-Einstein访问学者和CRTD / TU Dresden的Alexander von Humboldt教授获得支持。

        利益争夺

        作者声明没有竞争性的经济利益。

        伦理

        根据机构,国家和欧洲法规(批准号:APAFIS 3292-2015 1221 09359224和APAFIS 10545),按照马赛伦理委员会批准的方案,在无特定病原体的条件下进行体内程序。 -2017 0710 08253541)。

        参考

        1. DeFalco,T.,Potter,S.J。,Williams,A.V.,Waller,B.,Kan,M.J。和Capel,B。(2015)。 巨噬细胞对成人睾丸中的精原细胞生态位有贡献。 Cell Rep 12(7):1107-1119。
        2. Gautier,EL,Shay,T.,Miller,J.,Greter,M.,Jakubzick,C.,Ivanov,S.,Helft,J.,Chow,A.,Elpek,KG,Gordonov,S.,Mazloom, AR,Ma'ayan,A.,Chua,WJ,Hansen,TH,Turley,SJ,Merad,M.,Randolph,GJ和Immunological Genome,C。(2012)。 基因表达谱和转录调控途径,它们构成了小鼠组织巨噬细胞的特性和多样性的基础。 Nat Immunol 13(11):1118-1128。
        3. Gentek,R.,Molawi,K。和Sieweke,M。H.(2014)。 组织巨噬细胞的身份和自我更新。 Immunol Rev 262(1):56-73。
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引用:Mossadegh-Keller, N. and Sieweke, M. H. (2019). Characterization of Mouse Adult Testicular Macrophage Populations by Immunofluorescence Imaging and Flow Cytometry. Bio-protocol 9(5): e3178. DOI: 10.21769/BioProtoc.3178.
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