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The lumen of gastrointestinal tract is exposed to several potentially pathogenic microorganisms, thus it is extremely relevant to understand how immunosurveilance can be established. Peyer’s Patches (PPs) are oval or round lymphoid nodules that protrude from the outer wall of the ileum portion of small intestine. PPs contain a high percentage of B and T lymphocytes, macrophages and dendritic cells. Here we summarize a protocol for isolation and culture of mouse PP cells, which can be used to get a better insight into immunopathologies of microbes and to evaluate immune responses elicited by mucosal vaccines.

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Isolation and In vitro Activation of Mouse Peyer’s Patch Cells from Small Intestine Tissue
小鼠小肠派尔结的淋巴结细胞分离和体外激活

免疫学 > 免疫细胞分离 > 淋巴细胞
作者: Claudia Pastori
Claudia PastoriAffiliation: Division of Immunology, San Raffaele Scientific Institute, Milan, Italy
Bio-protocol author page: a1763
 and Lucia Lopalco
Lucia LopalcoAffiliation: Division of Immunology, San Raffaele Scientific Institute, Milan, Italy
For correspondence: lopalco.lucia@hsr.it
Bio-protocol author page: a1764
Vol 4, Iss 21, 11/5/2014, 4293 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1282

[Abstract] The lumen of gastrointestinal tract is exposed to several potentially pathogenic microorganisms, thus it is extremely relevant to understand how immunosurveilance can be established. Peyer’s Patches (PPs) are oval or round lymphoid nodules that protrude from the outer wall of the ileum portion of small intestine. PPs contain a high percentage of B and T lymphocytes, macrophages and dendritic cells. Here we summarize a protocol for isolation and culture of mouse PP cells, which can be used to get a better insight into immunopathologies of microbes and to evaluate immune responses elicited by mucosal vaccines.
Keywords: Mucosae(粘膜), Peyer's Patch(派伊尔结), Intestine(肠)

[Abstract]

Materials and Reagents

  1. Mouse (e.g. Balb/c) 8-20 weeks old
  2. Sterile PBS without Ca/Mg (Euroclone, catalog number: ECB4004L )
  3. Pen/Strep: 100x solution of penicillin/streptomycin/fungizone (Life Technologies, Gibco®, catalog number: 15240-062 )
  4. Gentamycin (50 mg/ml solution) (Life Technologies, Gibco®, catalog number: 15750-037 )
  5. RPMI 1640 (with glutamine) (Lonza, catalog number: BE12-702F )
  6. Fetal bovine serum (FBS) (heat inactivated) (Lonza, catalog number: DE14-801F )
  7. 0.4% trypan blue (Sigma-Aldrich, catalog number: T8154 )
  8. Phorbol 12-Myristate 13-Acetate (PMA) (Sigma-Aldrich, catalog number: P8139 - follow the manufacturer's instructions to prepare stock solutions)
  9. Ionomycin (Sigma-Aldrich, catalog number: I9657 - follow the manufacturer's instructions to prepare stock solutions)
  10. Common disinfectant (i.e. 70-90% ethanol, tincture of iodine)
  11. PBS + 2% Pen/Strep (see Recipes)
  12. RPMI + 2% Pen/Strep (see Recipes)
  13. Complete medium (see Recipes)

Equipment

  1. Soft wood tablet and pins
  2. Aluminium foils (or another smooth, non-absorbent surface)
  3. Three sterile small thin surgical scissors
    Note: In particular one for cutting the skin, the second one for cutting the muscular wall and the intestine, and the last one for removing PPs.
  4. Four sterile small thin surgical tweezers, two straight and two curved (straight ones for cutting the skin, the muscular wall and the intestine; curved ones to expel faeces and to remove PPs)
  5. 70 µm cell strainer (BD Biosciences, Falcon®, catalog number: 352350 )
  6. 2.5 ml syringes
  7. 15 and 50 ml centrifuge tubes
  8. 24-well cell culture plates
  9. Refrigerated centrifuge
  10. Sterile flow hood
  11. Thermostatic water bath
  12. Inverted microscope for cell cultures
  13. 37 °C, 5% CO2 cell culture incubator

Procedure


Figure 1. Diagram for the isolation of PP cells from small intestine. Modified from Lefrancois and Lycke (2001).

  1. Removal of small intestine and excision of Peyer’s patches
    1. Euthanize mouse by CO2 asphyxiation, following guide lines approved by your Institutional Animal Care and Use Committee. Proceed as soon as possible to the aseptic intestine explantation and PP dissociation to obtain maximum cell viability.
      Note: All subsequent steps should be performed in a sterile flow hood.
    2. Gently lay down the mouse on its back, on a soft wood surface, stretch the limbs and fix the four paws with pins.
    3. Clean the abdomen with the disinfectant.
    4. A pair of scissors and a straight tweezers are used to perform a midline incision and retract the skin, then open the muscular wall with another cutting along median axis with a new pair of scissors and tweezers.
    5. Cut small intestine approximately 0.5 cm below the stomach, draw out of peritoneal cavity unfolding it. Cut intestine about 1 cm above the caecum, then remove fat, mesenteric lymph nodes and adjacent tissues.
      Note: You can cut the intestine with the same scissors and tweezers used for the muscular wall.
    6. Place the intestine on an aluminium foil and continuously moisten with cold PBS + 2% Pen/Strep to prevent drying.
    7. Expel faeces by placing a curved tweezers flat on the surface of the intestine and press down along the entire length. Press gently to avoid breaking the intestine.
    8. Remove PPs by tightening the patch with a new curved tweezers and cutting it with scissors, avoiding as much as possible the surrounding intestinal wall. The patches appear as small protruding whitish/greyish nodules (diameter 1-2 mm), similar to the lymph nodes, embedded in the outer intestinal wall, which appears to be yellow-brown. In general, five to ten PPs can be obtained from a single mouse, depending of age and strain.
      Notes:
      1. The presence of an excessive amount of surrounding epithelium reduces the yield of recovered cells, as it could prevent a good mechanical dissociation.
      2.  The removal of the surrounding tissue is crucial if the enzymatic dissociation is carried out; in this last case the intraepithelial cells could contaminate the resulting PP-single cells.
    9. Immediately after excision, save all PPs in 1 ml cold RPMI + 2% Pen/Strep.

  2. Isolation of PP cells by mechanical dissociation
    1. Transfer all PPs in a 70 µm cell strainer placed over a 50 ml tube.
    2. Dissociate PPs using the plunger of a 2.5 ml syringe and gently crush the patches forcing through the filter. During dissociation wash the filter with 5 ml of cold RPMI + 2% Pen/Strep (medium flow through the filter contains the single cells of PPs).
    3. Repeat step 2 twice more, until they are no longer visible pieces of tissue. At this point, the tube contain PP-single cells flowed through the filter and suspended in 15 ml of RPMI + 2% Pen/Strep.
    4. Centrifuge at 400 x g, 4 °C for 10 min. Discard the supernatant, resuspend the cells and add 10 ml of cold RPMI + 2% Pen/Strep.
    5. Repeat step 4 two more times. After the last centrifugation resuspend the cells in 1-2 ml of cold RPMI + 2% Pen/Strep.
    6. Count viable cells using trypan blue dye exclusion.
      Notes:
      1. The variability in the number of cells recovered from a single mouse is very high, mainly due to the mouse (strain and age), the time elapsed between the euthanization and dissociation of PP, and the accuracy in removing every single PP.
      2. The mechanical dissociation allows to isolate mainly non-adherent cells, such as B and T lymphocytes. Adherent cells including monocytes, dendritic and non-immune epithelial cells appear to be present in small quantities since they require an enzymatic treatment to be isolated. The phenotype of recovered PP cells can be checked by FACS analysis.
      3. If needed, PP cells can be further purified by sorting with magnetic beads or FACS [see Guo et al. (2008) and Thornton (2003) in the reference section].

  3. In vitro activation of PP cells
    1. Centrifuge an appropriate amount of cells at 400 x g, at 4 °C for 10 min.
    2. Resuspend the pellet in complete medium at a concentration of 2 x 106 cells/ml and culture the PP cells in 24-well plate, 1 ml/well, in presence of 10 ng/ml PMA and 500 ng/ml Ionomycin for at least 24 h, in a 37 °C and 5% CO2 incubator.
      Note: Other activation protocols, specific for B or T lymphocytes, can be used [see Li et al. (2006) and Muul et al. (2011) in the references section].
      Optionals:
      1. To eliminate adherent cells, PP cells can be cultured for 2-4 h in Complete Medium (see recipes) with PMA and Ionomycin without antibiotics. The presence of adherent cells (monocytes and/or dendritic cells) may differentially modulate the activation of B and T lymphocytes with a consequent alterations of the immune responses.
      2. To avoid as much as possible a bacterial contamination, PP cells are washed and cultured for 24 h as describe above, in Complete Medium + PMA/Ionomycin, with 100 μg/ml Gentamycin instead of Penicillin/Streptomycin. The use of a different antibiotic ensures the elimination of all kinds of bacteria.
    3. After 24 h (or more) the supernatants of cultures can be collected, centrifuged at 400 x g, at 4 °C for 10 min to eliminate the cells, and used to test the presence of specific antibodies or cytokines. Activated cells can be harvested, washed by centrifugation at 4 °C for 10 min and used in flow cytometry assays and sorting.

Representative data

FACS analysis of in vitro activated PP cells.
PPs were obtained from 6 Balb/c mice as previously published (Pastori et al., 2014). In detail, two different immunization protocols were used: 3 mice received one intranasal (i. n.), while the other 3 mice received one intraperitoneal (i. p.) immunization. Aluminium hydroxide was used as adjuvant in i. p. immunization. For immunization Flock House Virus (FHV) was used in which an external loop of CCR5 (a seven transmembrane protein, belonging to chemokine receptor family and coreceptor for HIV-1) was introduced. The detailed immunization protocol has been described in Pastori et al. (2014).
After excision, PPs were mechanically dissociated and PP cells were in vitro activated with PMA and Ionomycin, as describe above. After 24 h cells were harvested and stained with antibodies against some markers of B cell activation.
As shown in Figure 2, CD138 and CD40/CD19 expression indicate a B cell activated phenotype for both protocols.


Figure 2. Expression of CD19, CD40, CD138 and co-expression of CD19/CD40 on PP cells after 24 h of PMA/Ionomycin activation. Percentage of positive cells refers to lymphocytes (gated on FSC vs SSC plot). Mean and standard deviation are shown.

Notes

  1. Images and more detailed information about mouse necropsy, intestine explantation and Peyer’ patches can be found in Scudamore (2013).
  2. The yield of cells isolated from PPs or intestinal lymph nodes is time sensitive. Proceed with the isolation as quickly as possible to obtain better cell viability.
  3. The mechanical dissociation of PPs gives a lower yield compared to the enzymatic one, but it does not alter the expression of surface antigens, thus, it is better in the case of subsequent FACS analysis or in vitro assays. The mechanical dissociation also leads to a relative loss of adherent accessory cells, such monocytes and/or dendritic cells, therefore, for the isolation of these cell types the enzymatic dissociation is preferable.

Recipes

Note: Prepare and keep all solutions in a sterile condition.

  1. PBS + 2% Pen/Strep
    For 100 ml combine 98 ml of PBS and 2 ml of Pen/Strep
    Refrigerate at 4 °C before use
  2. RPMI + 2% Pen/Strep
    For 100 ml combine 98 ml of RPMI and 2 ml of Pen/Strep
    Refrigerate at 4 °C before use
  3. Complete medium
    For 100 ml combine 89 ml of RPMI (with Glutamine), 10 ml of heat inactivated FBS and 1 ml of Pen/Strep
    Warm at 37 °C in a water bath before use

Acknowledgements

We thank Maria Rescigno for her help in mouse PPs isolation. This work was supported by Italian Ministry of Health, grant 40H15.

References

  1. Guo, Z., Jang, M. H., Otani, K., Bai, Z., Umemoto, E., Matsumoto, M., Nishiyama, M., Yamasaki, M., Ueha, S., Matsushima, K., Hirata, T. and Miyasaka, M. (2008). CD4+CD25+ regulatory T cells in the small intestinal lamina propria show an effector/memory phenotype. Int Immunol 20(3): 307-315.
  2. Lefrancois, L. and Lycke, N. (2001). Isolation of mouse small intestinal intraepithelial lymphocytes, Peyer's patch, and lamina propria cells. Curr Protoc Immunol Chapter 3: Unit 3 19.
  3. Li, Y. Y. Y., Yang, Y., Bao, M., Edwards III, C. K. and Parnes, J. R. (2006). Mouse splenic B lymphocyte activation using different activation stimuli induces in vitro splicing of tumor necrosis factor-α nuclear pre-mRNA. Mol Immunol 43(6): 613-622.
  4. Muul, L. M., Heine, G., Silvin, C., James, S. P., Candotti, F., Radbruch, A. and Worm, M. (2011). Measurement of proliferative responses of cultured lymphocytes. Curr Protoc Immunol Chapter 7: Unit7 10.
  5. Pastori, C., Diomede, L., Venuti, A., Fisher, G., Jarvik, J., Bomsel, M., Sanvito, F. and Lopalco, L. (2014). Induction of HIV-Blocking Anti-CCR5 IgA in Peyers's Patches without Histopathological Alterations. J Virol 88(7): 3623-3635.
  6. Scudamore, C. L. (2013). A Practical Guide to the Histology of the Mouse, John Wiley & Sons.
  7. Sheridan, B. S. and Lefrancois, L. (2012). Isolation of mouse lymphocytes from small intestine tissues. Curr Protoc Immunol Chapter 3: Unit 3 19.
  8. Thornton, A. M. (2003). Fractionation of T and B cells using magnetic beads. Curr Protoc Immunol 3.5 A. 1-3.5 A. 11.

材料和试剂

  1. 小鼠(例如 Balb/c)8-20周龄
  2. 无Ca/Mg的无菌PBS(Euroclone,目录号:ECB4004L)
  3. Pen/Strep:100x青霉素/链霉素/真菌的溶液(Life Technologies,Gibco ,目录号:15240-062)
  4. 庆大霉素(50mg/ml溶液)(Life Technologies,Gibco ,目录号:15750-037)
  5. RPMI 1640(含谷氨酰胺)(Lonza,目录号:BE12-702F)
  6. 胎牛血清(FBS)(热灭活的)(Lonza,目录号:DE14-801F)
  7. 0.4%台盼蓝(Sigma-Aldrich,目录号:T8154)
  8. Phorbol 12-肉豆蔻酸酯13-乙酸酯(PMA)(Sigma-Aldrich,目录号:P8139-按照制造商的说明制备储备溶液)
  9. 离子霉素(Sigma-Aldrich,目录号:I9657-按照制造商的说明书制备储备溶液)
  10. 常见的消毒剂( 70-90%乙醇,碘酊)
  11. PBS + 2%Pen/Strep(参见配方)
  12. RPMI + 2%Pen/Strep(参见配方)
  13. 完整介质(见配方)

设备

  1. 软木片和钉
  2. 铝箔(或另一个光滑,非吸收性表面)
  3. 三只无菌小手术剪刀
    注意:特别是一个用于切割皮肤,第二个用于切割肌肉的壁和肠,最后一个用于去除PP。
  4. 四个无菌小薄手术镊子,两个直和两个弯曲(直线切割皮肤,肌肉的墙和肠道;弯曲的排出粪便和移除PPs)
  5. 70μm细胞滤器(BD Biosciences,Falcon ,目录号:352350)
  6. 2.5 ml注射器
  7. 15和50ml离心管
  8. 24孔细胞培养板
  9. 冷冻离心机
  10. 无菌流量罩
  11. 恒温水浴
  12. 细胞培养的倒置显微镜
  13. 37℃,5%CO 2细胞培养箱中培养

程序


图1.从小肠分离PP细胞的图。从Lefrancois和Lycke(2001)修改。

  1. 去除小肠和切除淋巴集结
    1. 安乐死小鼠通过CO 2窒息,遵循的指导线 您的机构动物护理和使用委员会。 立即进行 可能无菌性肠道外植和PP解离 获得最大细胞活力 注意:所有后续步骤都应在无菌流罩中进行。
    2. 轻轻地将鼠标放在它的背部,在柔软的木材表面,伸展四肢,用针固定四个爪子。
    3. 用消毒剂清洁腹部。
    4. 一把剪刀和一个直镊子用于执行 中线切口和收缩皮肤,然后打开肌肉的墙 另一个剪切沿中轴与一副新的剪刀和 镊子
    5. 切小肠约0.5厘米以下 胃,拉出腹膜腔展开它。 切肠约   在盲肠上方1厘米,然后除去脂肪,肠系膜淋巴结和 相邻组织 注意:您可以使用与肌肉墙相同的剪刀和镊子切割肠。
    6. 将肠子放在铝箔上,连续用冷PBS + 2%Pen/Strep润湿,以防止干燥。
    7. 通过将弯曲的镊子平放在表面上排出粪便 肠道并沿整个长度压下。 轻按以避免 打破肠。
    8. 通过用a拧紧补丁来移除PP 新的弯曲的镊子和用剪刀切开,避免多少 可能的周围肠壁。 补丁显示为小 突出白色/灰色结节(直径1-2毫米),类似于 淋巴结,嵌入在外肠壁,这似乎是 黄棕色。 通常,可以从单个获得5至10个PP 小鼠,取决于年龄和应变 注意:
      1. 存在 过量的周围上皮降低了产量 回收的细胞,因为它可以防止良好的机械解离。
      2. 如果酶的,去除周围组织是至关重要的 进行解离; 在这最后一种情况下上皮内细胞 可能污染所得的PP-单细胞。
    9. 切除后,立即保存所有PP在1毫升冷RPMI + 2%钢笔/Strep。

  2. 通过机械解离分离PP细胞
    1. 将所有PP转移到放置在50ml管上的70μm细胞过滤器中
    2. 使用2.5毫升注射器柱塞轻轻粉碎解离PPs 补丁强制通过过滤器。 解离期间洗涤 用5ml冷RPMI + 2%Pen/Strep过滤(培养基流过 过滤器包含PP的单个单元格)。
    3. 重复步骤2两次 更多,直到它们不再是可见的组织片。 在此刻, 管含有PP-单细胞流过过滤器并悬浮   在15ml RPMI + 2%Pen/Strep中。
    4. 400℃离心4分钟,4℃   10分钟。 弃去上清液,重悬细胞并加入10 ml 冷RPMI + 2%Pen/Strep
    5. 重复步骤4两次。 最后一次离心后,将细胞重悬在1-2ml冷RPMI + 2%Pen/Strep中
    6. 使用台盼蓝染料排除计数活细胞 注意:
      1. 从单个小鼠中回收的细胞数目的变异性是   非常高,主要是由于老鼠(应变和年龄),经过的时间 PP的安乐死和解离之间的准确性 删除每个PP。
      2. 机械解离允许 主要分离非贴壁细胞,如B和T淋巴细胞。 附着   细胞包括单核细胞,树突和非免疫上皮细胞 似乎以少量存在,因为它们需要酶促的   治疗孤立。 回收PP细胞的表型可以是 通过FACS分析检查。
      3. 如果需要,可以进一步使用PP细胞 通过用磁珠或FACS分选进行纯化[参见Guo et al。 (2008) 和Thornton(2003)在参考部分]。

  3. 体外 PP细胞的活化
    1. 在4℃下,将合适量的细胞以400×g离心10分钟。
    2. 在完全培养基中以2×10 6个细胞/ml的浓度重悬沉淀物,并在24孔板中培养PP细胞,1ml /孔, 存在10ng/ml PMA和500ng/ml伊奥霉素至少24小时   37℃和5%CO 2培养箱 注意:其他激活协议, 对B或T淋巴细胞具有特异性[参见Li et al。 (2006)和 Muul et al。 (2011)in the references section]。
      可选:
      1. 到 消除贴壁细胞,PP细胞可以在Complete中培养2-4小时   中等(见配方)与PMA和没有抗生素的离子霉素。 的 粘附细胞(单核细胞和/或树突细胞)的存在 差异调节B和T淋巴细胞的激活 随后改变免疫应答。
      2. 尽量避免 尽可能细菌污染,洗涤和培养PP细胞 如上所述24小时,在完全培养基+ PMA /离子霉素中,用100   μg/ml庆大霉素代替青霉素/链霉素。 使用a 不同的抗生素确保消除各种细菌。
    3. 24小时(或更多)后,可以收集培养物的上清液, 在4℃下离心4分钟,10分钟以除去细胞 用于测试特异性抗体或细胞因子的存在。活性  可以收获细胞,通过在4℃下离心10分钟来洗涤  用于流式细胞术测定和分选。

代表数据

体外激活的PP细胞的FACS分析。
PPs从6 Balb/c小鼠获得,如先前公开的(Pastori等人,2014)。详细地,使用两种不同的免疫方案:3只小鼠接受一次鼻内(i.n.),而其它3只小鼠接受一次腹膜内(i.p.)免疫。氢氧化铝用作i。 p。免疫。对于免疫使用Flock House病毒(FHV),其中引入CCR5的外部环(7个跨膜蛋白,属于趋化因子受体家族和HIV-1的共同受体)。详细的免疫方案已在Pastori等人(2014)中描述。
切除后,将PP机械解离,并如上所述用PMA和离子霉素体外激活PP细胞。 24小时后收获细胞并用针对B细胞活化的一些标志物的抗体染色 如图2所示,CD138和CD40/CD19表达指示两种方案的B细胞活化表型。


图2.PMA /离子霉素活化24小时后CD19,CD40,CD138的表达和CD19/CD40在PP细胞上的共表达。阳性细胞的百分比是指淋巴细胞(在FSC对SSC情节)。显示了平均值和标准偏差。

笔记

  1. 关于小鼠尸检,肠移植和派克的图像和更详细的信息可以在Scudamore(2013)中找到。
  2. 从PP或肠淋巴结分离的细胞的产量是时间敏感的。尽可能快地进行分离以获得更好的细胞活力。
  3. 与酶相比,PP的机械解离产生较低的产量,但其不改变表面抗原的表达,因此,在随后的FACS分析或体外实验中更好。 机械解离还导致附着的附属细胞,例如单核细胞和/或树突细胞的相对损失,因此,为了分离这些细胞类型,酶解离是优选的。

食谱

注意:准备并保持所有溶液在无菌条件下。

  1. PBS + 2%Pen/Strep
    对于100ml,混合98ml PBS和2ml Pen/Strep
    使用前在4°C冷藏
  2. RPMI + 2%Pen/Strep
    对于100ml组合98ml RPMI和2ml Pen/Strep
    使用前在4°C冷藏
  3. 完成媒介
    对于100ml组合物,将89ml RPMI(含谷氨酰胺),10ml热失活的FBS和1ml Pen/Strep
    使用前在37°C水浴中温暖

致谢

我们感谢玛丽亚Rescigno她帮助鼠标PPs隔离。 这项工作是由意大利卫生部,支持40H15。

参考文献

  1. Guo,Z.,Jang,MH,Otani,K.,Bai,Z.,Umemoto,E.,Matsumoto,M.,Nishiyama,M.,Yamasaki,M.,Ueha,S.,Matsushima,K., ,T.and Miyasaka,M。(2008)。 CD4 + CD25 + 调节性T细胞在小肠固有层中显示效应子/记忆表型。 Int Immunol 20(3):307-315。
  2. Lefrancois,L.and Lycke,N。(2001)。 小鼠小肠上皮内淋巴细胞,淋巴集结和固有层细胞的分离。 em> Curr Protoc Immunol Chapter 3:Unit 3 19.
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How to cite this protocol: Pastori, C. and Lopalco, L. (2014). Isolation and In vitro Activation of Mouse Peyer’s Patch Cells from Small Intestine Tissue. Bio-protocol 4(21): e1282. DOI: 10.21769/BioProtoc.1282; Full Text



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