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Apr 2019
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Characterization of Immunological Niches within Peyer’s Patches by ex vivo Photoactivation and Flow Cytometry Analysis
体外光激活和流式细胞术分析派尔斑免疫龛位   

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Abstract

T follicular helper (Tfh) cells regulate B cell selection for entry into the germinal center (GC) reaction or for differentiation into antibody forming cells. This process takes place at the border between the T and B zones in lymphoid organs and involves physical contacts between T and B cells. During these interactions, T cells endow the B cells with selection signals that promote GC seeding or plasmablast differentiation based on their B cell receptor affinity. In Peyer’s patches (PPs), T cells promote B cell colonization of the subepithelial dome (SED) without effective affinity-based clonal selection. To specifically characterize the T cell population that resides within the SED niche, we performed ex vivo photoactivation of the SED compartment followed by flow cytometry analysis of the labeled cells, as described in this protocol. This technique integrates both spatial and cellular information in studies of immunological niches and can be adapted to various experimental systems.

Keywords: Photoactivation (光激活), Two-photon microscopy (双光子显微镜), SED (SED), Tfh cells (辅助性滤泡T细胞), Flow cytometry (流式细胞计数)

Background

Affinity maturation, the process wherein the affinity of serum antibodies towards a specific antigen increases over time, is achieved by selection of B cells bearing high affinity BCRs within germinal centers (GCs). Increase in antibody affinity is mediated through iterative cycles of somatic hyper mutation and affinity-based selection, a process which is orchestrated by T follicular helper (Tfh) cells (Kepler and Perelson, 1993; Oprea and Perelson, 1997; Victora and Nussenzweig, 2012). The GC is comprised of two microanatomical sites; the dark zone, where B cells proliferate and acquire somatic hypermutations, and the light zone, where B cells interact with cognate antigen and T cells. Although intravital imaging techniques were able to define immune cell dynamics in GCs (Allen et al., 2007; Hauser et al., 2007; Schwickert et al., 2007), transition between the GC zones remained in question. This problem was later solved by the generation of mice expressing photoactivatable GFP (PA-GFP) (Victora et al., 2010).

PA-GFP is a GFP variant whose peak excitation wavelength shifts from 415 nm (inactive PA-GFP) to 495 nm (active PA-GFP) upon two-photon irradiation at 830 nm. The non-activated PA-GFP is fluorescent as well, and this property can be used to distinguish between the photoactivated area and the total cells (Patterson and Lippincott-Schwartz, 2002).

The use of mice expressing PA-GFP provided direct evidence for interzonal migration in the GC and for the definition of the major GC exit zone (Victora et al., 2010; Stoler-Barak et al., 2019). Combination of intravital two-photon laser scanning microscopy with in situ photoactivation, allowed the microanatomical labeling of distinct niches within the germinal center, and led to the discovery that T cell help controls the movement between the two GC zones (Victora et al., 2010).

Cell-surface markers are commonly used to define a cell population in a specific niche; however, distinctive markers are not always available, generating a gap between the cellular and spatial information in the studied tissue. Furthermore, unknown cell populations that reside within a specific niche are usually hard to detect and characterize by conventional techniques. To overcome these limitations, photoactivation-based approaches have been used for unbiased identification of tissue-resident immune cells with minimal a priori knowledge of unique cell-surface marker expression. Niche-specific landmarks are often introduced into mice prior to labeling by photoactivation to define the area of interest. For example, adoptive cell transfer of fluorescently labeled B cells mark the B cell area within a tissue and can guide the selection of the region of interest for photoactivation (Medaglia et al., 2017). In the study associated with this protocol and as described here, we specifically photoactivated the subepithelial dome niche within the Peyer’s patch (Biram et al., 2019). This protocol can be adapted to other niches and additional tissues of interest.

Materials and Reagents

  1. Sterile Syringe 3 ml, luer lock (MedHarmony, catalog number: 181110 )
  2. Cell Strainer Nylon, Frame PP, pore size 70 μm, sterile (SPL Life Sciences, catalog number: 93070 )
  3. MonojectTM 18 G blunted cannula (Covidien, catalog number: 8881202348 )
  4. High precision microscope cover glasses 18 x 18 mm, 1.5H (Marienfeld, catalog number: 0107032 )
  5. Sandblasted single frosted pre-cleaned microscope slides, 25 x 75 mm x 1 mm thick (Thermo Fisher Scientific, catalog number: 421-004T )
  6. UBC PA-GFP mouse (The Jackson Laboratory, catalog number: 022486)
    This mouse strain carries a transgene of a photoactivatable variant of the GFP protein under the regulatory control of the constitutively expressed human ubiquitin C (UBC) promoter. Therefore, it enables rapid and stable fluorescent labeling of living cells.
  7. Calcium and Magnesium free phosphate buffered saline (PBS -/-) (Biological Industries, catalog number: 02-023-1A )
  8. Ethylenediaminetetraacetic acid solution (EDTA) (Sigma-Aldrich, catalog number: 03690)
  9. Fetal Bovine Serum, charcoal stripped (Thermo Fisher Scientific, catalog number: 12676029 )
  10. Silicone grease (can be found in hardware stores)
  11. Double distilled water (DDW)
  12. TruStain FcXTM (anti-mouse CD16/32) Antibody (Biolegend, clone: 93, catalog number: 101319 )
  13. Brilliant violet 605 anti-mouse B220 (CD45R) antibody (Biolegend, clone: RA3-6B2, catalog number: 103243 )
  14. APC-Alexa Fluor 750 anti-mouse CD4 antibody (Thermo Fisher Scientific, clone:S3.5, catalog number: MHCD0427 )
  15. PE anti-mouse CD44 antibody (Biolegend, clone: IM7, catalog number: 103023 )
  16. Alexa Fluor 700 anti-mouse CD62L antibody (Thermo Fisher Scientific, clone: MEL-14, catalog number: 56-0621-82 )
  17. PE/Cy7 anti-mouse CD279 (PD-1) antibody (Biolegend, clone: RMP1-30, catalog number: 109109 )
  18. Biotin anti-mouse CD185 (CXCR5) antibody (Biolegend, clone: L138D7, catalog number: 145509 )
  19. Alexa Fluor 647 Streptavidin (Biolegend, catalog number: 405237 )
  20. FACS buffer (see Recipes)

Equipment

  1. Tunable two-photon laser scanning microscope (TPLSM) equipped with a 20x water lens (Zeiss LSM 880 upright microscope fitted with Coherent Chameleon Vision laser)
  2. CytoFlex flow cytometer (Beckman Coulter)

Procedure

Note: Photoactivation is a time consuming process, therefore it is recommended to evaluate the time necessary for photoactivation of all required niches and analyze a sample size where samples will not wait for more than 3 h for the following staining step. Longer delay in tissue processing might increase the frequency of dead cells and therefore will not contribute to pool of analyzed cells. In the case of the SED photoactivation, ~30 SED regions were photoactivated in each mouse and up to two mice were analyzed a day. In addition, photoactivation can be held on the full PA-GFP mouse, on transferred cells or in chimeras prepared with PA-GFP bone marrow. Heterozygous PA-GFP mice can also be used in this protocol, but homozygous PA-GFP mice generate a stronger GFP signal. In the study associated with this protocol, we generated chimeric mice composed of 90% homozygous PA-GFP bone marrow and 10% AID-GFP bone marrow. AID is expressed by SED and GC B cells, thus AID-GFP was used as a landmark for these compartment.

  1. Euthanize a mouse by cervical dislocation or by CO2 inhalation (the technique does not affect the following steps).
  2. Open the abdominal cavity and remove the small intestine from the cecum to stomach into a PBS containing petri dish (~15 ml of PBS).
  3. Flush the intestine with 40 ml PBS using a 30 ml syringe with an 18 G MonojectTM blunted cannula. Avoid contact of the tissue with the intestinal content flushed out.
  4. Excise a piece of the intestine that includes a Peyer’s patch and cut it longitudinally without damaging the PP.
  5. Place the tissue on a cover slip glass and add silicone grease around the tissue. Make sure that the silicone layer is not thicker than the tissue.
  6. Add PBS (~30 μl) to hydrate the tissue and cover with an additional cover slip glass (Figure 1).


    Figure 1. Peyer’s patch slide preparation. A. Intestinal tissue containing a Peyer’s patch was cut out (black dashed lines) and an additional longitudinal cut (yellow dashed line) was performed to expose the SED compartment. B. The PP was placed on a cover slip, and hydrated with PBS bound by silicone grease.

  7. Place the tissue on a slide with the desired side for photoactivation facing up.
  8. Add a drop of DDW on the tissue and place the lens in the region of interest.
  9. Focus and image the tissue at 940 nm wavelength (this will also ensure that unspecific photoactivation will take place).
  10. Define a 5 µm interval Z-stack within the tissue in the area of desired photoactivation.
  11. Image a high quality Z-stack image before photoactivation at 940 nm (Figure 2).
  12. Define the area of photoactivation by adding a region in the ZEN setup and make sure to mark this region for acquisition.
  13. Change laser parameter to 830 nm and image. This should include imaging of a specific region of interest with a Z-stack at 830 nm.
  14. Return laser to 940 nm and remove the marked region.
  15. Repeat image acquisition at 940 nm (Figure 2). This should include the entire field with a Z-stack. The photoactivated area will appear green according to the defined region boundaries.


    Figure 2. Photoactivation of the SED area in a PP of a chimeric PA-GFP mouse. Images show the SED before photoactivation, during photoactivation at 830 nm and after photoactivation to indicate the labeled population.

  16. Repeat for all desired niches.
  17. Open cover slip glasses and gently transfer the tissue into a petri dish with a 70 µm cell strainer and 3 ml FACS buffer.
  18. Using a piston of a 3 ml syringe mash the PPs to create a single-cell suspension of PP-derived cells.
  19. Transfer the cell suspension into a FACS tube with a cell-strainer cap.
  20. Wash the petri dish with additional 1 ml of FACS buffer and collect the solution into the same FACS tube to maximize cell collection. The sample contains all PP cells, ranging 3-10 M cells.
  21. Spin down the cells at 300 RCF for 7 min at 4 °C.
  22. Discard the supernatant and resuspend the cells with the residual buffer (approximately 100 µl).
  23. Add 1 µl Fc blocker antibody (TruStain FcXTM), vortex and place on ice.
  24. Incubate 5 min on ice.
  25. Add 100 µl staining mix prepared in FACS buffer. In the paper associated with this protocol, Tfh cell markers were used to stain for SED-derived T cells (B220- CD4+ CD44+ CD62L- PD-1+ CXCR5+). In cases where a biotin-conjugated antibody is used, first stain the cells with the biotin-conjugated antibody together with other conjugated antibodies, and in a second step stain the cells with the streptavidin antibody. Since the residual volume in the tube following blocking is approximately 100 μl, the antibody mix is prepared as a 2x concentration to achieve final dilution as indicated in Table 1.

    Table 1. List of antibodies used for T cell staining in photoactivated PPs


  26. Vortex and incubate on ice for 30 min in the dark to allow surface marker staining.
  27. Wash the cells with 2 ml FACS buffer and spin down the cells at 300 RCF for 7 min at 4 °C.
  28. Add 100 µl staining mix containing 1:400 AF647 Streptavidin antibody (stock concentration: 0.5 mg/ml) prepared in FACS buffer.
  29. Vortex and incubate on ice for 30 min in the dark to allow surface marker staining.
  30. Wash the cells with 2 ml FACS buffer and spin down the cells at 300 RCF for 7 min at 4 °C.
  31. Resuspend the cells with 500 µl FACS buffer and analyze the cells using CytoFlex flow cytometer (Beckman Coulter).

Notes

  1. Since the frequency of the photoactivated cell population is very low (~1-5% of total single live cells), record as many events as possible to achieve a reliable number of cells for further analysis.
  2. In each experiment, extract and stain PPs derived from a mouse that was not photoactivated as a negative control. This control is crucial for proper gating on the photoactivated cell population.
    Gate on single live cells and out of this population gate on the V500+ GFP+ cell population. The V500+ represent the entire photoactivatable cell population while the cells that are GFP+ are the cells that were labeled by the two-photon microscope. This population can be analyzed for specific markers of interest. In the paper associated with this protocol we stained for Tfh cells as previously described (Liu, 2012).

Recipes

  1. FACS buffer
    Calcium and Magnesium free PBS (-/-)
    2% fetal bovine serum (FBS)
    1 mM EDTA
    To 500 ml PBS add 10 ml of PBS and 1 ml of 0.5 M EDTA
    Note: FACS buffer is kept up to one month at 4 °C.

Data analysis

As previously described, in the work associated with this study (Biram et al., 2019), chimeric mice were used to label the SED and GC compartments. Full flow cytometry analysis of T cell populations is available in the original paper. As shown in the representative plots in Figure 3, photoactivated cells were gated out of single lymphocytes and analyzed according to the markers of interest. In the case of photoactivation of non-chimeric mice, such as the UBC-PA GFP mouse, two populations will appear on the plot: all cells will be V500+ and only the photoactivated cells will appear as V500+ GFP+. When photoactivating a sample of transferred cells, a V500- population will represent the endogenous cell population. Photoactivated cell frequency is relatively low (~1-2%) and for appropriate statistical analysis, it is recommended to record a total of ~2 M cells.


Figure 3. Gating strategy for photoactivated-cells in mouse PPs. Representative flow cytometry plots showing the gating strategy on the PA-GFP cell population in PA-GFP AID-GFP bone marrow chimeras as in Biram et al. (2019). A. Live lymphocytes were gated as shown. B. Doublets were removed using the FSA-A/FSC-width distribution. C. V500 was used to mark all PA-GFP derived cells. Non-photoactivated cells (V500+ GFP-), AID-GFP- cells, AID-GFP+ cells, and photoactivated cells are represented as shown in the plot.

Acknowledgments

Z.S. is supported by the European Research Council (grant No. 677713), Human Frontiers of Science Program (grant No. CDA-00023/2016), Israel Science Foundation (grant No. 1090/18), Azrieli Foundation, Rising Tide Foundation and the Morris Kahn Institute for Human Immunology. Z.S. is a member in the European Molecular Biology Organization Young Investigator Program and is supported by grants from The Benoziyo Endowment Fund for the Advancement of Science, The Sir Charles Clore Research Prize, Comisaroff Family Trust, Irma & Jacques Ber-Lehmsdorf Foundation, Gerald O. Mann Charitable Foundation and David M. Polen Charitable Trust.
  This protocol provides details regarding photoactivation and flow cytometry analysis of Peyer’s patch niches as previously described (Biram et al., 2019).

Competing interests

The author declare no competing interests.

Ethics

All experimental procedures have been approved by the Weizmann Institute Animal Care and Use Committee (IACUC) and followed all relevant ethical regulations, IACUC number 00960118-4.

References

  1. Allen, C. D., Okada, T., Tang, H. L. and Cyster, J. G. (2007). Imaging of germinal center selection events during affinity maturation. Science 315(5811): 528-531.
  2. Biram, A., Strömberg, A., Winter, E., Stoler-Barak, L., Salomon, R., Addadi, Y., Dahan, R., Yaari, G., Bemark, M. and Shulman, Z. (2019). BCR affinity differentially regulates colonization of the subepithelial dome and infiltration into germinal centers within Peyer's patches. Nat Immunol 20(4): 482-492.
  3. Hauser, A. E., Junt, T., Mempel, T. R., Sneddon, M. W., Kleinstein, S. H., Henrickson, S. E., von Andrian, U. H., Shlomchik, M. J. and Haberman, A. M. (2007). Definition of germinal-center B cell migration in vivo reveals predominant intrazonal circulation patterns. Immunity 26(5): 655-667.
  4. Kepler, T. B. and Perelson, A. S. (1993). Cyclic re-entry of germinal center B cells and the efficiency of affinity maturation. Immunol Today 14(8): 412-415.
  5. Liu, Z. (2012). FACS staining for follicular helper T cells. Bio-protocol 2(2): e35.
  6. Medaglia, C., Giladi, A., Stoler-Barak, L., De Giovanni, M., Salame, T. M., Biram, A., David, E., Li, H., Iannacone, M., Shulman, Z. and Amit, I. (2017). Spatial reconstruction of immune niches by combining photoactivatable reporters and scRNA-seq. Science 358(6370): 1622-1626.
  7. Oprea, M. and Perelson, A. S. (1997). Somatic mutation leads to efficient affinity maturation when centrocytes recycle back to centroblasts. J Immunol 158(11): 5155-5162.
  8. Patterson, G. H. and Lippincott-Schwartz, J. (2002). A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297(5588): 1873-1877.
  9. Schwickert, T. A., Lindquist, R. L., Shakhar, G., Livshits, G., Skokos, D., Kosco-Vilbois, M. H., Dustin, M. L. and Nussenzweig, M. C. (2007). In vivo imaging of germinal centres reveals a dynamic open structure. Nature 446(7131): 83-87.
  10. Stoler-Barak, L., Biram, A., Davidzohn, N., Addadi, Y., Golani, O. and Shulman, Z. (2019). B cell dissemination patterns during the germinal center reaction revealed by whole-organ imaging. J Exp Med 216(11): 2515-2530.
  11. Victora, G. D. and Nussenzweig, M. C. (2012). Germinal centers. Annu Rev Immunol 30: 429-457.
  12. Victora, G. D., Schwickert, T. A., Fooksman, D. R., Kamphorst, A. O., Meyer-Hermann, M., Dustin, M. L. and Nussenzweig, M. C. (2010). Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter. Cell 143(4): 592-605.

简介

[摘要] T卵泡辅助细胞(Tfh )调节B细胞的选择,使其进入生发中心(GC)反应或分化为抗体形成细胞。此过程发生在淋巴器官的T和B区之间的边界,涉及T和B细胞之间的物理接触。在这些相互作用中,T细胞赋予B细胞选择信号,这些信号根据其B细胞受体亲和力促进GC接种或成浆细胞分化。在Peyer氏斑(PPs)中,T细胞在没有有效亲和力的情况下促进上皮下穹sub (SED)的B细胞定殖。 ed克隆选择。为了具体表征驻留在SED利基空间中的T细胞群体,我们按照本方案中所述对SED隔室进行了离体光激活,然后进行了标记细胞的流式细胞仪分析。该技术将空间和细胞信息整合到了免疫小生境研究中,可以适应各种实验系统。

【背景技术】亲和力成熟是血清抗体对特定抗原的亲和力随时间而增加的过程,这是通过在生发中心(GC)中选择带有高亲和力BCR的B细胞来实现的。增加抗体亲和力通过体细胞超突变和基于亲和力的选择,这由T滤泡辅助(编排的处理的反复循环介导的TFH )细胞奥普雷亚;(开普勒和Perelson,1993 和Perelson,1997; Victora和Nussenzweig, 2012)。GC由两个微观解剖部位组成;暗区(B细胞增殖并获得体细胞超突变)和亮区(B细胞与相关抗原和T细胞相互作用)。尽管活体成像技术能够确定GC中的免疫细胞动力学(Allen 等,2007; Hauser 等,2007; Schwickert 等,2007),但GC区之间的过渡仍存在问题。该问题随后通过表达光可活化的GFP(PA-GFP)的小鼠的产生而得以解决(Victora 等,2010)。

PA-GFP是一种GFP变体,在830 nm处的双光子照射下,其峰值激发波长从415 nm(无效PA-GFP)变为495 nm(有效PA-GFP)。非活化的PA-GFP也具有荧光,并且该性质可以用于区分光活化的区域和总细胞(Patterson和Lippincott-Schwartz,2002)。

表达PA-GFP的小鼠的使用为GC中的区域间迁移和主要GC出口区的定义提供了直接证据(Victora 等,2010; Stoler-Barak 等,2019)。活体双光子激光扫描显微镜与原位光激活相结合,可以在生发中心内对不同的壁ches 进行显微解剖标记,并导致发现T细胞有助于控制两个GC区之间的运动(Victora 等,2010)。 )。

细胞表面标记通常用于定义特定位置的细胞群。然而,独特的标记并不总是可用的,从而在被研究组织中的细胞信息和空间信息之间产生了差距。此外,驻留在特定位置的未知细胞群通常很难通过常规技术检测和表征。为了克服这些局限性,基于光激活的方法已被用于组织驻留免疫细胞的无偏鉴定,而对独特的细胞表面标志物表达的先验知识却很少。在通过光激活进行标记以定义目标区域之前,通常将利基特定的地标引入小鼠。例如,荧光标记的B细胞的过继细胞转移标记了组织内的B细胞区域,并可以指导目标区域的选择以进行光激活(Medaglia 等,2017)。在与此协议相关的研究中,如本文所述,我们专门激活了Peyer斑块内的上皮下穹n生态位(Biram et al。,2019)。该协议可以适用于其他利基和其他感兴趣的组织。

关键字:光激活, 双光子显微镜, SED, 辅助性滤泡T细胞, 流式细胞计数

材料和试剂


 


无菌注射器3 ml,鲁尔锁(MedHarmony ,目录号181110)
细胞过滤网尼龙,PP框架,孔径70 μ米,无菌(SPL生命科学,目录号:93070)
Monoject TM 18 G钝插管(Covidien,目录号:8881202348)
高精度显微镜盖玻片18 x 18 mm,1.5H(Marienfeld ,目录号:0107032)
25 x 75 mm x 1 mm厚的喷砂单磨砂预清洁显微镜载玻片(Thermo Fisher Scientific,目录号:421-004T)
UBC PA-GFP小鼠(杰克逊实验室,目录号:022486)
该小鼠品系在组成型表达的人泛素C(UBC)启动子的调控下携带GFP蛋白可光活化变体的转基因。因此,它能够快速,稳定地对活细胞进行荧光标记。


不含钙和镁的磷酸盐缓冲盐水(PBS -/- )(Biological Industries,目录号:02-023-1A)
乙二胺四乙酸溶液(EDTA)(Sigma - Aldrich,目录号:03690)
胎牛血清,去除木炭(Thermo Fisher Scientific,目录号:12676029)
硅脂(可以在五金店找到)
双蒸馏水(DDW)
TruStain FcX TM (抗小鼠CD16 / 32)抗体(Biolegend ,克隆:93,目录号:101319)
艳紫色605抗小鼠B220(CD45R)抗体(Biolegend ,克隆:RA3-6B2,目录号:103243)
APC-Alexa Fluor 750抗小鼠CD4抗体(Thermo Fisher Scientific,克隆:S3.5,目录号:MHCD0427)
PE抗小鼠CD44抗体(Biolegend ,克隆:IM7,目录号:103023)
Alexa Fluor 700抗小鼠CD62L抗体(Thermo Fisher Scientific,克隆:MEL-14,目录号:56-0621-82)
PE / Cy7抗小鼠CD279(PD-1)抗体(Biolegend ,克隆:RMP1-30,目录号:109109)
生物素抗小鼠CD185(CXCR5)抗体(Biolegend ,克隆:L138D7,目录号:145509)
Alexa Fluor 647链霉亲和素(Biolegend ,目录号:405237)
FACS缓冲区(s 配方)
 


设备


 


可调光子激光扫描显微镜(TPLSM),配备20倍水透镜(Zeiss LSM 880垂直显微镜,配备相干变色龙视觉激光)
CytoFlex 流式细胞仪(贝克曼库尔特公司)
 


程序


 


注意:光活化是一个耗时的过程,因此建议评估所有必需位的光活化所需的时间,并分析样品大小,样品在接下来的染色步骤中等待时间不会超过3小时。组织处理中的较长延迟可能会增加死细胞的频率,因此不会有助于分析细胞的聚集。在SED光激活的情况下,每只小鼠中约30个SED区域被光激活,每天最多分析两只小鼠。此外,可以在完整的PA-GFP小鼠,转移的细胞或用PA-GFP骨髓制备的嵌合体中进行光激活。杂合PA-GFP小鼠也可用于该方案,但纯合PA-GFP小鼠产生更强的GFP信号。在与此协议相关的研究中,我们生成了由90%纯合PA-GFP骨髓和10%AID-GFP骨髓组成的嵌合小鼠。AID由SED和GC B细胞表达,因此AID-GFP被用作这些区室的标志。


 


通过颈脱位或CO 2 吸入使小鼠安乐死(该技术不会影响以下步骤)。
打开腹腔,将盲肠的小肠移至胃中,放入含有培养皿的PBS(约15 ml PBS)中。
使用装有18 G Monoject TM 钝头套管的30 ml注射器,用40 ml PBS冲洗肠道。避免将肠内容物冲洗掉而与组织接触。
切下一个带有Peyer贴片的小肠,然后纵向切割它,而不会损坏PP。
将薄纸放在盖玻片上,并在薄纸周围添加硅脂。确保硅胶层不厚于薄纸。
加入PBS(〜30 μ 升)以水合组织和盖有附加盖玻片玻璃(图1)。
 


D:\ Reformatting \ 2020-2-7 \ 1902741--1321 Adi Biram 847099 \ Figs jpg \ Fig1.jpg


图1. Peyer的贴片玻片制备。A. 切下含有派伊尔氏淋巴集结的肠组织(黑色虚线),并进行另外的纵向切割(黄色虚线)以暴露SED隔室。B. 钍Ë PP放置在盖玻片和水合,用PBS结合硅脂。


 


将组织放在一张幻灯片上,使所需的光激活面朝上。
在组织上滴一滴DDW,然后将晶状体放在目标区域。
在940 nm波长处聚焦并成像组织(这还将确保发生非特异性的光激活)。
在所需光激活区域内的组织内,定义一个5 µm间隔的Z-stack。
在940 nm进行光激活之前,对高质量的Z堆栈图像进行成像(图2)。
通过在ZEN设置中添加区域来定义光激活区域,并确保标记该区域以进行采集。
将激光参数更改为830 nm和图像。这应包括在830 nm处使用Z堆栈对特定的特定区域成像。
将激光返回940 nm,然后移动标记的区域。
在940 nm处重复图像采集(图2)。这应该包括带有Z堆栈的整个字段。根据定义的区域边界,光激活区域将显示为绿色。
 


D:\ Reformatting \ 2020-2-7 \ 1902741--1321 Adi Biram 847099 \ Figs jpg \ Fig2.jpg


图2.嵌合PA-GFP小鼠PP中SED区域的光活化。图像显示了在光激活之前,在830 nm的光激活期间以及在光激活之后的SED,以指示标记的群体。


 


对所有需要的位置重复上述步骤。
操作盖上盖玻片,并用70 µm细胞过滤器和3 ml FACS缓冲液将组织轻轻转移到培养皿中。
使用3毫升注射器的活塞捣碎PP,以产生PP衍生细胞的单细胞悬液。
将细胞悬液转移到带有细胞滤网盖的FACS管中。
用另外的1 ml FACS缓冲液洗涤培养皿,并将溶液收集到同一FACS管中以最大程度地收集细胞。样品包含所有PP细胞,范围为3-10 M细胞。
在4°C下以300 RCF旋转细胞7分钟。
丢弃上清液,并用残留的缓冲液(约100 µl)重悬细胞。
加入1 µl Fc阻断剂抗体(TruStain FcX TM ),涡旋并置于冰上。
在冰上孵育5分钟。
加入在FACS缓冲液中制备的100 µl染色混合物。在与此协议相关的论文中,使用Tfh 细胞标记物对SED衍生的T细胞(B220 - CD4 + CD44 + CD62L - PD-1 + CXCR5 + )进行染色。在使用生物素结合抗体的情况下,首先用生物素结合抗体与其他结合抗体一起对细胞进行染色,然后在第二步中用链霉亲和素抗体对细胞进行染色。由于在下面的阻挡管中的剩余体积为约100 μ 升,抗体混合物被制备成2倍浓度,以实现最终稀释度为指示Ť 能够1 。




表1. 用于光活化PP中T细胞染色的抗体列表


记号笔


最终稀释


原料浓度[mg / ml]


μ 克/ 100 μ 升混合物(2×)


BV605 B220


1:400


0.2


0.1


APC-AF750 CD4


1:400


0.2


0.1


PE CD44


1:400


0.2


0.1


AF700 CD62L


1:400


0.5


0.25


PE \ Cy7 PD-1


1:600


0.2


0.067


生物素CXCR5


1:100


0.5


1个


 


涡旋并在黑暗中于冰上孵育30分钟,以使表面标记物染色。
用2 ml FACS缓冲液洗涤细胞,并在300 RCF和4°C下旋转细胞7分钟。
加入在FACS缓冲液中制备的含有1:400 AF647链霉亲和素抗体(储备浓度:0.5 mg / ml)的100 µl染色混合物。
涡旋并在黑暗中于冰上孵育30分钟,以使表面标记物染色。
用2 ml FACS缓冲液洗涤细胞,并在300 RCF和4°C下旋转细胞7分钟。
用500 µl FACS缓冲液重悬细胞,并使用CytoFlex 流式细胞仪(Beckman Coulter)分析细胞。
 


笔记


 


由于光活化细胞的频率非常低(约占单个活细胞总数的1-5%),因此请记录尽可能多的事件,以获取可靠数目的细胞用于进一步分析。
在每个实验中,提取和染色来自未光活化作为阴性对照的小鼠的PPs 。这种控制对于正确激活光活化细胞群至关重要。
在单个活细胞上进行门操作,并在V500 + GFP + 细胞群体上进行此门操作。V500 + 代表整个可光激活的细胞群,而GFP + 的细胞则是由双光子显微镜标记的细胞。可以分析该人群的特定目标标记。在与该方案相关的论文中,我们按照先前的描述对Tfh 细胞进行了染色(Liu,2012)。


 


菜谱


 


FACS缓冲区
不含钙和镁的PBS(-/-)


2%胎牛血清(FBS)


1毫米EDTA


向500 ml PBS中加入10 ml PBS和1 ml 0.5 M EDTA


注意:FACS缓冲液在4 °C下最多可保存一个月。


数据分析


 


如前所述,在与这项研究相关的工作中(Biram et al。,2019),使用嵌合小鼠标记SED和GC间隔。原始论文提供了T细胞群体的全流式细胞术分析。如图3中的代表性图所示,将光活化细胞选出单个淋巴细胞,并根据目标标记进行分析。如果是非嵌合小鼠(例如UBC-PA GFP小鼠)的光激活,则该图中将出现两个种群:所有细胞均为V500 + ,只有光激活的细胞显示为V500 + GFP + 。当光活化转移细胞的一个样品,V500 - 人口将代表内源性的细胞群。光活化细胞的频率相对较低(〜1-2%),为了进行适当的统计分析,建议记录总共约2 M个细胞。


 


D:\ Reformatting \ 2020-2-7 \ 1902741--1321 Adi Biram 847099 \ Figs jpg \ Fig3.jpg


图3 。小鼠PP中光活化细胞的门控策略。代表性的流式细胞术图显示了Biram 等人在PA-GFP AID-GFP骨髓嵌合体中PA-GFP细胞群体的门控策略。(2019)。A. 现场淋巴细胞门如图所示。B. d oublets使用的FSA-A / FSC-宽度分布去除。C. V500用于标记所有PA-GFP衍生的细胞。非光活化细胞(V500 + GFP - ),AID-GFP - 细胞,AID-GFP + 细胞,和光活化的细胞被表示为显示在曲线图。


 


致谢


 


ZS是由欧洲研究委员会(批准号:677713),科学计划的人类前沿的支持(批准号:CDA-00023/2016),以色列科学基金会(批准号:一零九零年至1018年),阿兹列里基金会,涨潮基金会莫里斯·卡恩人体免疫研究所。ZS是欧洲分子生物学组织年轻研究者计划的成员,并获得了来自Benoziyo 科学促进基金会,查尔斯· 克洛爵士爵士研究奖,科米萨罗夫家庭信托基金,艾玛与雅克· 伯勒姆斯多夫基金会,杰拉尔德·奥的资助。曼恩慈善基金会和大卫· 波伦慈善信托基金。


  如先前所述(Biram et al。,2019),该协议提供了有关Peyer斑的光激活和流式细胞仪分析的详细信息。


利益争夺


 


作者宣称没有利益冲突。


 


伦理


 


所有实验程序均已获得魏茨曼研究所动物护理和使用委员会(IACUC)的批准,并遵循所有相关的道德法规(IACUC编号00960118-4)。


 


参考文献


 


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引用:Biram, A. and Shulman, Z. (2020). Characterization of Immunological Niches within Peyer’s Patches by ex vivo Photoactivation and Flow Cytometry Analysis. Bio-protocol 10(6): e3562. DOI: 10.21769/BioProtoc.3562.
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