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Identification of Helminth-induced Type 2 CD4+ T Cells and ILC2s
鉴定蠕虫诱导产生的2型 CD4+T细胞和第2组先天淋巴细胞(ILC2)的方法

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Abstract

After activation, T cells differentiate into different T helper (Th) subsets, namely Th1, Th2, and Th17. These different Th subsets are associated with the production of particular cytokines endowing them with different functions. In immunity against helminth infections the Th2 cell subset plays an important role. Th2 cells typically produce IL-4, IL-5, IL-13, and IL-9 resulting in antibody-isotype switching to IgE, eosinophilia, basophilia, mucin production, and smooth muscle cell hyperactivity. Here we analyze the development of the pathogen specific Th2 immune responses in mice after infection with the helminth parasite, Heligmosomoides polygyrus bakeri, the induction of innate lymphoid cells type 2 (ILC2) and the activation of the inflammasome in macrophages by excretory/secretory products of Heligmosomoides polygyrus bakeri.

Part I. Generation and collection of Heligmosomoides poylgyrus (Hp) excretory/secretory (ES) products (HES)

Materials and Reagents

  1. Adult L5 Heligmosomoides poylgyrus (Hp)
  2. 50 ml PBS (Life Technologies, Gibco®, catalog number: 10010-015 )
  3. Complete RPMI (GE Healthcare, catalog number: E15-840 )
  4. 10,000 MWCO cellulose membrane (EMD Millipore, Centriprep®)
  5. EndoTrap Blue 5/1 LPS-binding affinity column (Hyglos GmbH, catalog number: 311063 )
  6. HES wash solution I (see Recipes)
  7. HES wash solution II (see Recipes)
  8. HES culturing solution (see Recipes)

Equipment

  1. Aluminum foil
  2. 50 ml Falcon tube (BD, catalog number: 352070 )
  3. Pipette
  4. Falcon flask (TPP Techno Plastic Products, catalog number: 90076 )
  5. Nanodrop (Thermo Fisher Scientific)
  6. Centrifuge (Eppendorf, model: 5810R )
  7. Perti dish
  8. Microscope (OLYMPUS, model: CKX31 )
  9. Laminar airflow cabinet (Faster Ultrasafe, model: US218D )

Procedure

  1. Work sterile under using a laminar airflow cabinet.
  2. Wash adult L5 Hp extensively in sterile 50 ml PBS in a 50 ml Falcon tube.
  3. Mix by inverting the Falcon flask. Wait 1-5 min until L5 Hp sink to the bottom of the flask, then remove as much as possible of the supernatant by using a pipette.
  4. Repeat steps 1-2 for 3x.
  5. Transfer L5 Hp into 50 ml wash solution I as it was previously described by Camberis et al. (2003).
  6. Mix by inverting the Falcon tube. Wait 1-5 min until L5 Hp sink to the bottom of the flask, then remove as much as possible of the supernatant.
  7. Repeat steps 4-5 for 3x.
  8. Transfer settled L5 Hp into new 50 ml Falcon tube using a 10 ml pipette and gently suck the worms from the bottom of the 50 ml tube and fill up with to 50 ml with wash solution II.
  9. Mix by inverting and incubate 1 h at RT.
  10. Count L5 Hp worms by transfering to a perti dish using a dissecting microscope.
  11. Transfer settled L5 Hp worms into an appropriate cell culture flask at 100 worms/ml in HES culturing solution.
  12. Incubate at 37 °C in cell culture isolator with 5% CO2.
  13. Collected supernatant every 2 day using a pipette for a period of 2 weeks, followed by sterile filtration and concentration of the supernatant by centrifugation through a 10,000 MWCO cellulose membrane using a swinging bucket rotor at 4,000 x g, 25 °C, and a starting volume of 15 ml. Refresh the medium in the worm culture flask with the same volume of culturing solution. Measure the protein concentration in the concentrated HES using a NanoDrop.
  14. Remove LPS contamination from HES culture supernatants using an EndoTrap Blue LPS-binding affinity column according to the manufacturer instructions.

Recipes

  1. HES wash solution I
    500 ml PBS
    2 ml gentamycin reagent solution (50 mg/ml)
    10 ml 100x penicillin-streptomycin solution (10,000 U/ml)
  2. HES wash solution II
    500 ml RPMI 1640
    2 ml gentamycin reagent solution (50 mg/ml)
    10 ml 100x penicillin-streptomycin solution (10,000 U/ml)
  3. HES culturing solution
    500 ml RPMI 1640
    1 ml gentamycin reagent solution (50 mg/ml)
    5 ml 100x penicillin-streptomycin solution (10,000 U/ml)
    50 ml 10% glucose

Part II. Identification of helminth-induced type 2 CD4+ T cells

Materials and Reagents

  1. Mesenteric lymph nodes (MLNs) from Hp infected mice
  2. Trypan blue (Life Technologies, Gibco®, catalog number: 15250-061 )
  3. Complete RPMI (GE Healthcare, catalog number: E15-840 )
  4. PMA (phorbol myristate acetate) (Sigma-Aldrich, catalog number: P8139 )
  5. Ionomycin (Sigma-Aldrich, catalog number: I9657 )
  6. Brefeldin A (Sigma-Aldrich, catalog number: B7651 )
  7. PBS (Life Technologies, Gibco®, catalog number: 10010-015 )
  8. BSA (Roche Diagnostics, catalog number: 10735108001 )
  9. Live /dead fixable aqua dead cell stain kit (Life Technologies, InvitrogenTM, catalog number: L34957 )
  10. PFA (Sigma-Aldrich, catalog number: P6148 )
  11. Saponin (Sigma-Aldrich, catalog number: 47036 )
  12. EDTA (AppliChem GmbH, catalog number: A3145 )
  13. FACS buffer (see Recipes)

Equipment

  1. Aluminum foil
  2. 70 μm cell strainer (Thermo Fisher Scientific, catalog number: 22363548 )
  3. 50 ml Falcon tube (BD, catalog number: 352070 )
  4. Neubauer counting chamber (Assistent®)
  5. Incubator (Thermo Fisher Scientific, HeracellTM 150i )
  6. 96 well cell culture plates (Corning, Costar®, catalog number: 3799 )
  7. Centrifuge (Eppendorf, model: 5810R )
  8. Microscope (OLYMPUS, model: CKX31 )
  9. BD LSR II flow cytometer (BD Biosciences)

Procedure

  1. Work on ice and under sterile conditions.
  2. Remove mesenteric lymph nodes (MLNs) from Hp infected mice and place in complete RPMI on ice (make sure to remove all fat tissue of the MLNs before you store them in complete RPMI).
  3. Smash MLNs trough a 70 μm cell strainer placed on an 50 ml Falcon tube and rinse with 20 ml complete RPMI.
  4. Count live cells using trypan blue and a hemacytometer (Neubauer counting chamber) in smashed MLN cell suspensions.
  5. Incubate MLN cell suspensions at 5 x 105 live cells per 200 μl complete RPMI supplemented with 5 μg/ml HES in round bottom 96 cell culture well plates in triplicates at 37 °C with 5% CO2.
  6. Leave cells for 72 h in the incubator.
  7. Centrifuge 96 well plates at 400 x g for 5 min at 4 °C.
  8. Discard supernatant (or freeze on -20 °C for further cytokine analysis).
  9. Resuspend cells in 100 μl complete RPMI supplemented with PMA (10-7 M) and Ionomycin (1 μg/ml).
  10. Incubate for additional 2 h at 37 °C with 5% CO2.
  11. Add 100 μl of complete RPMI supplemented with 20 μg/ml Brefeldin A.
  12. Incubate for additional 2 h at 37 °C with 5% CO2.
  13. Centrifuge 96 well plates at 400 x g for 5 min at 4 °C.
  14. Combine triplicates into a single well by resuspending one after the other with the same 200 μl PBS with 0.2% BSA.
  15. Centrifuge at 400 x g for 5 min at 4 °C.
  16. Resuspend with 200 μl PBS with 0.2% BSA.
  17. Centrifuge at 400 x g for 5 min at 4 °C.
  18. Resuspend with 50 μl/well 24G2 as FcR block (obtained from cell line culture supernatants) (1:800 dilution from stock 1 mg/ml stock solution) for 10 min.
  19. Centrifuge at 400 x g for 5 min at 4 °C and resuspend in 200 μl PBS with 0.5% BSA.
  20. Centrifuge at 400 x g for 5 min at 4 °C and resuspend in 100 μl PBS with 0.5% BSA supplemented with live /dead fixable aqua dead cell stain kit, according to the manufacturer instructions.
  21. Centrifuge at 400 x g for 5 min at 4 °C and resuspend in 200 μl PBS with 0.5% BSA.
  22. Centrifuge at 400 x g for 5 min at 4 °C and resuspend in 50 μl PBS with 0.5% BSA including the cell surface antibodies labeled with for FACS analysis.
  23. Incubate in the dark, e.g. cover with aluminum foil for 30 min.
  24. Centrifuge at 400 x g for 5 min at 4 °C and resuspend in 200 μl PBS with 0.5% BSA.
  25. Repeat step 24.
  26. Centrifuge at 400 x g for 5 min at 4 °C and resuspend in PBS with 2% PFA for 20 min for fixation.
  27. Centrifuge at 400 x g for 5 min at 4 °C and resuspend in 200 μl PBS with 0.5% BSA.
  28. Repeat step 27.
  29. Centrifuge at 400 x g for 5 min at 4 °C. Discard supernatant after centrifugation and resuspend in 100 μl saponin (10x diluted in PBS with 0.2% BSA) to permeabilise cells.
  30. Centrifuge at 400 x g for 5 min at 4 °C. Discard supernatant after centrifugation and resuspend in 50 μl saponin supplemented with intracellular cytokine antibodies labeled with fluorochrom for FACS analysis.
  31. Incubate in the dark, e.g. cover with aluminum foil for 40 min.
  32. Centrifuge at 400 x g for 5 min at 4 °C. Discard supernatant after centrifugation and resuspend in 200 μl saponin.
  33. Repeat step 32.
  34. Centrifuge at 400 x g for 5 min at 4 °C. Discard supernatant after centrifugation and resuspend in 200 μl PBS with 0.5% BSA.
  35. Repeat step 34.
  36. Centrifuge at 400 x g for 5 min at 4 °C. Discard supernatant after centrifugation and resuspend in 75 μl FACS buffer.
  37. Filter cells in a 70 μm cell strainer.
  38. Acquire cells at the LSRII.

Recipes

  1. FACS buffer
    500 ml PBS
    25 ml BSA (10%)
    20 ml EDTA (20 mM)

Part III. Identification of helminth-induced ILC2s

Materials and Reagents

  1. Mesenteric lymph nodes (MLNs) and spleens from Hp infected mice
  2. Complete RPMI (GE Healthcare, catalog number: E15-840 )
  3. PBS (Life Technologies, Gibco®, catalog number: 10010-015 )
  4. BSA (Roche Diagnostics, catalog number: 10735108001 )
  5. Live /dead fixable aqua dead cell stain kit (Life Technologies, InvitrogenTM, catalog number: L34957 )
  6. Biotin labeled cell surface antibodies (Biolegend)
    1. CD3 (clone: 145-2C11 )
    2. CD4 (clone: GK1.5 )
    3. CD11b (clone: M1/70 )
    4. CD11c (clone: N418 )
    5. CD19 (clone: 6D5 )
    6. Nk1.1 (clone: PK136 )
    7. Ly6G (clone: 1A8 )
    8. IgE (clone: RME-1 )
  7. C-kit (CD117) Pacific Blue (clone: 2B8 ) (Biolegend)
  8. sca-1 (Ly-6A/E) PE-Cy7 (clone: D7) (eBioscience)
  9. T1/ST2 FITC (clone: DJ8) (MD Bioproducts)
  10. ICOS (CD278) PerCp-Cy5.5 (clone: C398.4A ) (Biolegend)
  11. Streptavidin PE-TexasRed (BD Biosciences)
  12. FACS buffer (see Recipes)

Equipment

  1. 70 μm cell strainer
  2. 50 ml Falcon tube
  3. 3 ml syringe plunger
  4. 96 well plates
  5. BD LSR II flow cytometer (BD Biosciences)

Procedure

  1. Work on ice.
  2. Remove mesenteric lymph nodes (MLNs) and spleens from Hp infected mice and place them in complete RPMI on ice (make sure to remove all fat tissue of the MLNs and spleens before you store them in complete RPMI).
  3. Smash MLNs and spleens trough a 70 μm cell strainer placed on an 50 ml Falcon tube by using a 3 ml syringe plunger and rinse with 20 ml complete RPMI.
  4. Count live cells in smashed cell suspensions.
  5. Transfer up to 1 x 106 cells in 200 μl PBS with 0.2% BSA in a single round bottom 96 well.
  6. Centrifuge at 400 x g for 5 min at 4 °C. Discard supernatant after centrifugation.
  7. Resuspend with 200 μl PBS with 0.2% BSA.
  8. Centrifuge at 400 x g for 5 min at 4 °C. Discard supernatant after centrifugation.
  9. Resuspend with 50 μl/well 24G2 (1:800 dilution from stock 1 mg/ml stock solution) for 10 min.
  10. Centrifuge at 400 x g for 5 min at 4 °C. Discard supernatant after centrifugation and resuspend in 200 μl PBS with 0.5% BSA.
  11. Centrifuge at 400 x g for 5 min at 4 °C and resuspend in 100 μl PBS with 0.5% BSA supplemented with live /dead fixable aqua dead cell stain kit, according to the manufacturer instructions.
  12. Incubate in the dark, e.g. cover with aluminum foil for 20 min.
  13. Centrifuge at 400 x g for 5 min at 4 °C. Discard supernatant after centrifugation and resuspend in 200 μl PBS with 0.5% BSA.
  14. Centrifuge at 400 x g for 5 min at 4 °C. Discard supernatant after centrifugation and resuspend in 50 μl PBS with 0.5% BSA including the biotin labeled cell surface antibodies (CD3, CD4, CD11b, CD11c, CD19, Nk1.1, Ly6G and IgE) for the lineage negative gating and c-kit (Pacific Blue), sca-1 (PE-Cy7), T1/ST2 (FITC), ICOS (PerCp/Cy5.5).
  15. Incubate in the dark, e.g. cover with aluminum foil for 30 min.
  16. Centrifuge at 400 x g for 5 min at 4 °C. Discard supernatant after centrifugation and resuspend in 200 μl PBS with 0.5% BSA.
  17. Centrifuge at 400 x g for 5 min at 4 °C. Discard supernatant after centrifugation and resuspend in 50 μl PBS with 0.5% BSA including the streptavidin Texas red labeled antibody.
  18. Incubate in the dark, e.g. cover with aluminum foil for 30 min.
  19. Centrifuge at 400 x g for 5 min at 4 °C. Discard supernatant after centrifugation and resuspend in 200 μl PBS with 0.5% BSA.
  20. Centrifuge at 400 x g for 5 min at 4 °C. Discard supernatant after centrifugation and resuspend in 50 μl PBS with 2% PFA for 20 min for fixation.
  21. Centrifuge at 400 x g for 5 min at 4 °C. Discard supernatant after centrifugation and resuspend in 200 μl PBS with 0.5% BSA.
  22. Repeat step 20.
  23. Centrifuge at 400 x g for 5 min at 4 °C. Discard supernatant after centrifugation and resuspend in 75 μl FACS buffer.
  24. Filter cells in a 70 μm cell strainer.
  25. Acquire cells at the LSRII.
  26. Gating strategy is shown in Figure 1:


    Figure 1. Gating strategy used to identify ILC2s. ILC2 cells were identified as lineage-negative (Gr-1, CD3, CD19), Sca-1, c-kit, T1/ST2 and ICOS positive live cells present in the spleen of mice at 6 days post infection with the helminth parasite Heligmosomoides polygyrus. Y-axis represents counts if not otherwise indicated.

Recipes

  1. FACS buffer
    500 ml PBS
    25 ml BSA (10%)
    20 ml EDTA (20 mM)

Acknowledgments

This protocol is an extended version of the one described in Zaiss et al. (2013). The research has received funding from the European community seventh framework program [FP7/2009–2014] under EC-GA no[241642] and part of this work was funded by grants from the Swiss National Science Foundation and the Institute of Arthritis Research. KMM is supported by EMBO long-term fellowship and the Australian NHMRC post-doctoral fellowship.

References

  1. Camberis, M., Le Gros, G. and Urban, J., Jr. (2003). Animal model of Nippostrongylus brasiliensis and Heligmosomoides polygyrus. Curr Protoc Immunol Chapter 19: Unit 19 12.
  2. Zaiss, M. M., Maslowski, K. M., Mosconi, I., Guenat, N., Marsland, B. J. and Harris, N. L. (2013). IL-1beta suppresses innate IL-25 and IL-33 production and maintains helminth chronicity. PLoS Pathog 9(8): e1003531.

简介

活化后,T细胞分化成不同的T辅助(Th)子集,即Th1,Th2和Th17。 这些不同的Th亚群与赋予它们不同功能的特定细胞因子的产生相关。 在抵抗蠕虫感染的免疫中,Th2细胞亚群起重要作用。 Th2细胞通常产生IL-4,IL-5,IL-13和IL-9,导致抗体同种型转换为IgE,嗜酸性粒细胞增多,嗜碱性粒细胞,粘蛋白产生和平滑肌细胞多动。 在这里我们分析感染后的蠕虫寄生虫,Heligmosomoides polygyrus bakeri,诱导的先天淋巴细胞2型(ILC2)和炎症小体的激活的病原体特异性Th2免疫反应的发展 巨噬细胞通过 Heligmosomoides polygyrus bakeri的排泄/分泌产物。

(Hp)排泄/分泌(ES)产物(HES)的产生和收集

材料和试剂

  1. 成人L5 Heligmosomoides poylgyrus (Hp)
  2. 50ml PBS(Life Technologies,Gibco ,目录号:10010-015)
  3. 完全RPMI(GE Healthcare,目录号:E15-840)
  4. 10,000 MWCO纤维素膜(EMD Millipore,Centriprep )
  5. EndoTrap Blue 5/1 LPS结合亲和柱(Hyglos GmbH,目录号:311063)
  6. HES洗涤溶液I(参见配方)
  7. HES洗涤溶液II(参见配方)
  8. HES培养溶液(参见配方)

设备

  1. 铝箔
  2. 50ml Falcon管(BD,目录号:352070)
  3. 移液器
  4. Falcon烧瓶(TPP Techno Plastic Products,目录号:90076)
  5. Nanodrop(Thermo Fisher Scientific)
  6. 离心机(Eppendorf,型号:5810R)
  7. 佩尔蒂菜
  8. 显微镜(OLYMPUS,型号:CKX31)
  9. 层流气流柜(更快的Ultrasafe,型号:US218D)

程序

  1. 在使用层流气流柜的情况下工作无菌
  2. 在50ml Falcon管中,在无菌的50ml PBS中大量洗涤成体L5 Hp
  3. 通过倒置Falcon瓶混合。 等待1-5分钟,直到L5 Hp沉到烧瓶底部,然后使用移液管除去尽可能多的上清液。
  4. 对3x重复步骤1-2。
  5. 将L5 Hp转移到50mL洗涤溶液I中,如先前Camberis等人(2003)所述。
  6. 通过翻转Falcon管混合。 等待1-5分钟,直到L5 Hp沉到烧瓶底部,然后尽可能多地除去上清液
  7. 对3x重复步骤4-5。
  8. 转移沉降L5 Hp到新的50毫升Falcon管使用10毫升吸管,轻轻吸取蠕虫的50毫升管的底部,充满50毫升与洗涤溶液II。
  9. 通过倒置混合,并在室温下孵育1小时
  10. 计数L5 Hp蠕虫通过使用解剖显微镜转移到perti菜
  11. 将转移沉降的L5 Hp蠕虫在HES培养溶液中以100蠕虫/ml转移到合适的细胞培养瓶中。
  12. 在37℃下在具有5%CO 2的细胞培养隔离器中孵育
  13. 每2天使用移液管收集上清液,持续2周,然后无菌过滤,并使用摇摆转子在4,000×g,25°下通过10,000MWCO纤维素膜离心浓缩上清液 C,起始体积为15ml。 用等体积的培养溶液更新蜗杆培养瓶中的培养基。 使用NanoDrop测量浓缩HES中的蛋白质浓度
  14. 根据制造商的说明,使用EndoTrap Blue LPS结合亲和柱从HES培养物上清液中去除LPS污染物。

食谱

  1. HES洗涤溶液I
    500 ml PBS
    2ml庆大霉素试剂溶液(50mg/ml) 10ml 100×青霉素 - 链霉素溶液(10,000U/ml)
  2. HES洗液II
    500ml RPMI 1640
    2ml庆大霉素试剂溶液(50mg/ml) 10ml 100×青霉素 - 链霉素溶液(10,000U/ml)
  3. HES培养液
    500ml RPMI 1640
    1ml庆大霉素试剂溶液(50mg/ml) 5ml 100x青霉素 - 链霉素溶液(10,000U/ml) 50ml 10%葡萄糖

第二部分。 鉴定蠕虫诱导的2型CD4 + T细胞

材料和试剂

  1. 来自Hp感染的小鼠的肠系膜淋巴结(MLN)
  2. 台盼蓝(Life Technologies,Gibco ,目录号:15250-061)
  3. 完全RPMI(GE Healthcare,目录号:E15-840)
  4. PMA(佛波脂肉豆蔻酸乙酸酯)(Sigma-Aldrich,目录号:P8139)
  5. 离子霉素(Sigma-Aldrich,目录号:I9657)
  6. 布雷菲德菌素A(Sigma-Aldrich,目录号:B7651)
  7. PBS(Life Technologies,Gibco ,目录号:10010-015)
  8. BSA(Roche Diagnostics,目录号:10735108001)
  9. 活/死可固定水性死细胞染色试剂盒(Life Technologies,Invitrogen TM ,目录号:L34957)
  10. PFA(Sigma-Aldrich,目录号:P6148)
  11. 皂苷(Sigma-Aldrich,目录号:47036)
  12. EDTA(AppliChem GmbH,目录号:A3145)
  13. FACS缓冲区(参见配方)

设备

  1. 铝箔
  2. 70μm细胞过滤器(Thermo Fisher Scientific,目录号:22363548)
  3. 50ml Falcon管(BD,目录号:352070)
  4. Neubauer计数室(Assistent ®
  5. 孵育器(Thermo Fisher Scientific,Heracell TM 150i)
  6. 96孔细胞培养板(Corning,Costar ,目录号:3799)
  7. 离心机(Eppendorf,型号:5810R)
  8. 显微镜(OLYMPUS,型号:CKX31)
  9. BD LSR II流式细胞仪(BD Biosciences)

程序

  1. 在冰和无菌条件下工作。
  2. 从Hp感染的小鼠中取出肠系膜淋巴结(MLN),并置于完全的RPMI在冰上(确保删除MLN的所有脂肪组织,然后将其存储在完整的RPMI)。
  3. 粉碎MLN通过放置在50ml Falcon管上的70μm细胞过滤器并用20ml完全RPMI冲洗。
  4. 使用台盼蓝和血细胞计数器(Neubauer计数室)在捣碎的MLN细胞悬浮液中计数活细胞。
  5. 在37℃,5%CO 2 。
  6. 在培养箱中保持细胞72小时。
  7. 在4℃下将96孔板在400×g离心5分钟
  8. 弃去上清液(或在-20℃冷冻用于进一步的细胞因子分析)
  9. 将细胞重悬于补充有PMA(10μM至10μM)和离子霉素(1μg/ml)的100μl完全RPMI中。
  10. 在37℃下用5%CO 2孵育另外2小时
  11. 加入100μl补充有20μg/ml布雷菲德菌素A的完整RPMI
  12. 在37℃下用5%CO 2孵育另外2小时
  13. 在4℃下将96孔板在400×g离心5分钟
  14. 通过使用含有0.2%BSA的相同200μlPBS一个接一个地重悬,将三份重复混合到单个孔中。
  15. 在4℃下以400×g离心5分钟。
  16. 用含0.2%BSA的200μlPBS重悬
  17. 在4℃下以400×g离心5分钟。
  18. 用50μl/孔24G2作为FcR阻断(从细胞系培养物上清液获得)(来自贮备液1mg/ml储备溶液的1:800稀释液)重悬10分钟。
  19. 在4℃下以400×g离心5分钟,并重悬于200μl含有0.5%BSA的PBS中。
  20. 在4℃下以400×g离心5分钟,并根据制造商的说明书重悬于100μl含有补充有活/死可固定水性死细胞染色试剂盒的0.5%BSA的PBS中。
  21. 在4℃下以400×g离心5分钟,并重悬于200μl含有0.5%BSA的PBS中。
  22. 在4℃下以400×g离心5分钟,并重悬于含有0.5%BSA的50μlPBS中,包括标记有用于FACS分析的细胞表面抗体。
  23. 在黑暗中孵育,例如。 用铝箔覆盖30分钟。
  24. 在4℃下以400×g离心5分钟,并重悬于200μl含有0.5%BSA的PBS中。
  25. 重复步骤24.
  26. 在4℃下以400×g离心5分钟,并在含有2%PFA的PBS中重悬20分钟以固定。
  27. 在4℃下以400×g离心5分钟,并重悬于200μl含有0.5%BSA的PBS中。
  28. 重复步骤27.
  29. 在4℃下以400×g离心5分钟。 离心后弃去上清液,并重悬于100μl皂苷(用含0.2%BSA的PBS稀释10倍)中以透化细胞。
  30. 在4℃下以400×g离心5分钟。 离心后弃去上清液,并重悬于50μl添加有用荧光标记的细胞内细胞因子抗体的皂苷中,用于FACS分析。
  31. 在黑暗中孵育,例如用铝箔覆盖40分钟。
  32. 在4℃下以400×g离心5分钟。 离心后弃去上清液,并重悬于200μl皂苷中
  33. 重复步骤32.
  34. 在4℃下以400×g离心5分钟。 离心后弃去上清液,并重悬于200μl含0.5%BSA的PBS中
  35. 重复步骤34.
  36. 在4℃下以400×g离心5分钟。 离心后弃去上清液并重悬于75μlFACS缓冲液中
  37. 在70μm细胞过滤器中过滤细胞
  38. 在LSRII获取单元格。

食谱

  1. FACS缓冲区
    500 ml PBS
    25 ml BSA(10%) 20ml EDTA(20mM)

第三部分。 鉴定蠕虫诱导的ILC2s

材料和试剂

  1. 肠系膜淋巴结(MLN)和来自Hp感染的小鼠的脾
  2. 完全RPMI(GE Healthcare,目录号:E15-840)
  3. PBS(Life Technologies,Gibco ,目录号:10010-015)
  4. BSA(Roche Diagnostics,目录号:10735108001)
  5. 活/死可固定水性死细胞染色试剂盒(Life Technologies,Invitrogen TM ,目录号:L34957)
  6. 生物素标记的细胞表面抗体(Biolegend)
    1. CD3(克隆:145-2C11)
    2. CD4(克隆:GK1.5)
    3. CD11b(克隆:M1/70)
    4. CD11c(克隆:N418)
    5. CD19(克隆:6D5)
    6. Nk1.1(克隆:PK136)
    7. Ly6G(克隆:1A8)
    8. IgE(克隆:RME-1)
  7. C-kit(CD117)Pacific Blue(克隆:2B8)(Biolegend)
  8. sca-1(Ly-6A/E)PE-Cy7(克隆:D7)(eBioscience)
  9. T1/ST2 FITC(克隆:DJ8)(MD Bioproducts)
  10. ICOS(CD278)PerCp-Cy5.5(克隆:C398.4A)(Biolegend)
  11. 链霉亲和素PE-TexasRed(BD Biosciences)
  12. FACS缓冲区(参见配方)

设备

  1. 70μm细胞过滤器
  2. 50ml Falcon管
  3. 3 ml注射器柱塞
  4. 96孔板
  5. BD LSR II流式细胞仪(BD Biosciences)

程序

  1. 在冰上工作。
  2. 从Hp感染的小鼠中取出肠系膜淋巴结(MLN)和脾脏,将其置于冰上的完全RPMI中(确保在将它们储存在完全RPMI中之前除去MLN和脾脏的所有脂肪组织)。
  3. 粉碎MLN和脾通过使用3ml注射器柱塞并置于50ml Falcon管上的70μm细胞过滤器,并用20ml完全RPMI冲洗。
  4. 在捣碎的细胞悬浮液中计数活细胞
  5. 在单个圆底96孔中,在含有0.2%BSA的200μlPBS中转移至1×10 6个细胞。
  6. 在4℃下以400×g离心5分钟。 离心后弃去上清液。
  7. 用含0.2%BSA的200μlPBS重悬
  8. 在4℃下以400×g离心5分钟。 离心后弃去上清液。
  9. 用50μl/孔24G2(来自原液1mg/ml储备溶液的1:800稀释液)重悬10分钟。
  10. 在4℃下以400×g离心5分钟。 离心后弃去上清液,并重悬于200μl含0.5%BSA的PBS中
  11. 在4℃下以400×g离心5分钟,并根据制造商的说明书重悬于100μl含有补充有活/死可固定水性死细胞染色试剂盒的0.5%BSA的PBS中。
    在单个圆底96孔中,在含有0.2%BSA的200μlPBS中转移至1×10 6个细胞。
  12. 在4℃下以400×g离心5分钟。 离心后弃去上清液。
  13. 用含0.2%BSA的200μlPBS重悬
  14. 在4℃下以400×g离心5分钟。 离心后弃去上清液。
  15. 用50μl/孔24G2(来自原液1mg/ml储备溶液的1:800稀释液)重悬10分钟。
  16. 在4℃下以400×g离心5分钟。 离心后弃去上清液,并重悬于200μl含0.5%BSA的PBS中
  17. 在4℃下以400×g离心5分钟,并根据制造商的说明书重悬于100μl含有补充有活/死可固定水性死细胞染色试剂盒的0.5%BSA的PBS中。
    ...
  18. Incubate in the dark, e.g. cover with aluminum foil for 30 min.
  19. Centrifuge at 400 x g for 5 min at 4 °C. Discard supernatant after centrifugation and resuspend in 200 μl PBS with 0.5% BSA.
  20. Centrifuge at 400 x g for 5 min at 4 °C. Discard supernatant after centrifugation and resuspend in 50 μl PBS with 2% PFA for 20 min for fixation.
  21. Centrifuge at 400 x g for 5 min at 4 °C. Discard supernatant after centrifugation and resuspend in 200 μl PBS with 0.5% BSA.
  22. Repeat step 20.
  23. Centrifuge at 400 x g for 5 min at 4 °C. Discard supernatant after centrifugation and resuspend in 75 μl FACS buffer.
  24. Filter cells in a 70 μm cell strainer.
  25. Acquire cells at the LSRII.
  26. Gating strategy is shown in Figure 1:


    Figure 1. Gating strategy used to identify ILC2s. ILC2 cells were identified as lineage-negative (Gr-1, CD3, CD19), Sca-1, c-kit, T1/ST2 and ICOS positive live cells present in the spleen of mice at 6 days post infection with the helminth parasite Heligmosomoides polygyrus. Y-axis represents counts if not otherwise indicated.

Recipes

  1. FACS缓冲区
    500 ml PBS
    25 ml BSA(10%) 20ml EDTA(20mM)

致谢

该协议是Zaiss等人(2013)中描述的扩展版本。 研究获得了欧洲共同体第七框架计划[FP7/2009-2014]在EC-GA no [241642]下的资助,这项工作的一部分由瑞士国家科学基金会和关节炎研究所资助。 KMM由EMBO长期研究金和澳大利亚NHMRC博士后研究金支持。

参考文献

  1. Camberis,M.,Le Gros,G。和Urban,J.,Jr。(2003)。 巴西尼罗河线虫动物模型和 Heligmosomoides polygyrus >。 Curr Protoc Immunol Chapter 19:Unit 19 12.
  2. Zaiss,M.M.,Maslowski,K.M.,Mosconi,I.,Guenat,N.,Marsland,B.J.and Harris,N.L。(2013)。 IL-1beta抑制先天性IL-25和IL-33的产生,并维持蠕虫的长期性。 PLoS Pathog 9(8):e1003531。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Zaiss, M. M. and Maslowski, K. M. (2014). Identification of Helminth-induced Type 2 CD4+ T Cells and ILC2s. Bio-protocol 4(11): e1141. DOI: 10.21769/BioProtoc.1141.
  2. Zaiss, M. M., Maslowski, K. M., Mosconi, I., Guenat, N., Marsland, B. J. and Harris, N. L. (2013). IL-1beta suppresses innate IL-25 and IL-33 production and maintains helminth chronicity. PLoS Pathog 9(8): e1003531.
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