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Isolation and Analysis of Stromal Cell Populations from Mouse Lymph Nodes
小鼠淋巴结基质细胞群的分离和分析   

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Abstract

Our protocol describes a simple procedure for isolating stromal cells from lymph nodes (LN). LN are disrupted then enzymatically digested with collagenase and dispase to produce a single cell suspension that can be stained with fluorescently labelled antibodies and analysed by flow cytometry. This protocol will enable identification of fibroblastic reticular cells (FRC), lymphatic endothelial cells (LEC), blood endothelial cells (BEC) as PNAd+ BEC that form LN high endothelial venules (HEV). This method can be applied to examine LN stromal cell responses during inflammatory events induced by infections or immunologic adjuvants and to subset most leukocytes found in LN.

Keywords: Lymphoid stromal cells(淋巴基质细胞), Lymph nodes(淋巴结), Endothelial cells(内皮细胞), Fibroblastic reticular cells(成纤维网状细胞), Immune response(免疫反应), Virus infection(病毒感染)

Background

Lymph nodes (LN) are constructed of complex networks of mesenchymal and endothelial stromal cells. These include the fibroblastic reticular cells (FRCs), lymphatic endothelial cells (LECs) and blood endothelial cells (BECs). These stromal cells organize the complex microarchitecture of LN, enabling the support of immune cell migration, homeostasis, tolerance and cellular interactions required for the initiation of immune responses to pathogens and tumors. We have shown that the LN stromal cells can proliferate and expand in response to inflammatory signals and the recruitment of immune cells into LN that accompanies infections (Gregory et al., 2017). These stromal cells can also significantly modulate their transcriptional program to respond to infection, thereby supporting ongoing immune responses. This protocol enables reliable isolation of stromal cell subsets from LN both in the steady state and during disease. This enables phenotypic, functional, genetic or epigenetic investigation of LN stromal cells to reveal how they contribute to tissue homeostasis and immune responses.

Materials and Reagents

  1. 1,000 μl pipette tips (Neptune, catalog number: BT1250 )
  2. 200 μl pipette tips (Neptune, catalog number: BT200 )
  3. 20 μl pipette tips (Neptune, catalog number: BT20 )
  4. 10 μl pipette tips (Neptune, catalog number: BT10XLS3 )
  5. 24-well plate (Corning, catalog number: 3527 )
  6. 10 ml tubes (SARSTEDT, catalog number: 62.9924.284 )
  7. 50 ml tubes (Greiner Bio One International, catalog number: 227261 )
  8. Nylon mesh 70 μm (Clear Edge, catalog number: PA75-1750 )
  9. 5 ml polystyrene round bottom tube (Corning, Falcon®, catalog number: 352008 )
  10. 96-well round-bottom plate (Corning, catalog number: 3788 )
  11. Spatula (VWR, catalog number: 10806-408 )
  12. C57BL\6 mice (THE JACKSON LABORATORY, catalog number: 000664 )
  13. Blank calibration particles (BD, BD Biosciences, catalog number: 556296 )
  14. HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 11344033 )
  15. L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 21051024 )
  16. Benzylpenicillin (CSL, catalog number: AUST R 10329 )
  17. Streptomycin sulphate (Thermo Fisher Scientific, GibcoTM, catalog number: 11860038 )
  18. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  19. RPMI (Thermo Fisher Scientific, GibcoTM, catalog number: 11875093 )
  20. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10099141 )
  21. Collagenase D (Sigma-Aldrich, catalog number: 11088882001 )
    Manufacturer: Roche Diagnostics, catalog number: 11088882001 .
  22. Dispase II (Sigma-Aldrich, catalog number: 04942078001 )
    Manufacturer: Roche Diagnostics, catalog number: 04942078001 .
  23. DNase I (Sigma-Aldrich, catalog number: DN25-1G )
  24. Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  25. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906-500G )
  26. Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) (Sigma-Aldrich, catalog number: E5134-1KG )
  27. Antibodies
    1. gp38-PE, 8.1.1 (BioLgend, catalog number: 127408 )
    2. CD31-PE-Cy7, 390 (BioLegend, catalog number: 102524 )
    3. CD45.2-A700, 104 (BioLegend, catalog number: 109822 )
    4. Ter-119-A700, TER-119 (BioLegend, catalog number: 116220 )
    5. HEV-A488, MECA-79 (Thermo Fisher Scientific, eBioscienceTM, catalog number: 53-6036-82 )
    6. CD16/32-purified, 2.4G2 (BD, BD Biosciences, catalog number: 553142 )
  28. Live/Dead Fixable Near-IR cell stain kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: L10119 )
  29. Supplementum Completum (SC) (see Recipes)
  30. RP-2 solution (see Recipes)
  31. Digestion mix 1 (see Recipes)
  32. Digestion mix 2 (see Recipes)
  33. FACS buffer (see Recipes)
  34. Lymph node stromal cell antibody cocktail (see Recipes)

Equipment

  1. Water bath (VWR, catalog number: 89501-464 )
  2. Dumont No.5 forceps (Fine Science Tools, catalog number: 11251-10 )
  3. Graefe forceps (Roboz Surgical Instrument, catalog number: RS-5137 )
  4. Analytical balance for weighing (Ohaus, catalog number: 30208454 )
  5. 1,000 μl pipette (Eppendorf, catalog number: 3121000120 )
  6. 200 μl pipette (Eppendorf, catalog number: 3121000082 )
  7. 20 μl pipette (Eppendorf, catalog number: 3121000040 )
  8. 10 μl pipette (Eppendorf, catalog number: 3121000015 )
  9. Refrigerated centrifuge with plate carriers (Beckman Coulter, model: Allegra® X-15R , catalog number: B09822; Beckman Coulter, catalog number: 368914 )
  10. Flow cytometer (BD, BD Biosciences, model: BD LSRFORTESSA )

Software

  1. FlowJo (FlowJo)
  2. Prism (GraphPad)

Procedure

  1. Isolation and digestion of lymph nodes
    1. Prepare digestion mixes 1 and 2 (see Recipes) and place in a water bath at 37 °C.
      Note: The volume of the digestion mixes prepared is dependent on the number of samples required for the experiment. 1 ml of each digestion mix is required for up to 6 skin draining lymph nodes (LN). Digestion mixes are prepared fresh for each experiment. We prepare the required amount of each enzyme for each experiment. However, users can prepare a 100x stock of the enzymes (in RPMI) and store them at -20 °C for further usage if preferred.
    2. Harvest LN from mice, place in wells of a 24-well plate containing 1 ml of RP-2 solution per well (see Recipes) and keep on ice.
      Note: We typically process two skin draining LN from non-infected mice (inguinal, axillary or brachial). During an inflammatory episode such as Herpes Simplex Virus (HSV) flank infection, we process the brachial LN individually. If popliteal LN are isolated, we pool and process at least 4 LN from non-infected mice, and only one during an inflammatory event.
      To assist with the identification of mouse LN, users can refer to a diagram of the localization of LN in the mouse model, available here:
      http://www.informatics.jax.org/greenbook/figures/figure13-4.shtml
    3. Take the LN out of the plate and disrupt the LN into 4-5 pieces with two thin forceps.
    4. Transfer disrupted LN into a 10 ml tube and add 1 ml of digest mix 1.
      Note: During this step, LN are not put back into the 24-well plate. Thus they are digested in 1 ml with the digestion mix 1.
    5. Place tubes in the water bath and incubate for 25 min at 37 °C.
    6. Invert tube and flick a few times during digestion.
    7. After incubation, cut off the tip of 1 ml pipette and thoroughly mix the digested LN for 30 sec, up to 1 min. Leave undisturbed until remaining visible pieces of tissue settle to the bottom of the tube.
    8. Remove supernatant without taking the undigested pieces of LN and transfer into a 50 ml tube containing 15 ml of FACS buffer (see Recipes) and keep on ice.
      Note: Make sure to remove as much supernatant as possible without taking any pieces of LN. There should be minimal medium left in the tube. One round of digestion is not enough to properly digest LN and thus two rounds of digestion are necessary to maximize the yield of SC to be released.
    9. Add 1 ml of digestion mix 2 to the undigested pieces of LN left from step A7 and place in the water bath for 15 min at 37 °C.
    10. After incubation, thoroughly mix the digested LN with 1 ml pipette for 1 min.
    11. Transfer supernatant to tubes containing the cell suspension from step A8.
      Note: LN should completely be digested after this step. If any pieces are remaining, repeat steps A9 and A10 with 10-min incubation only.
    12. Centrifuge cell suspension for 5 min at 500 x g, 4 °C.
    13. Aspirate supernatant and resuspend cell suspension in 500 (for up to 6 skin draining LN, or popliteal LN) or 1,000 μl of FACS buffer (if more than 6 skin draining LN).
    14. Filter cell suspension through 70 μm nylon mesh in a 5 ml FACS tube and count cells.
      Note: Users can also use counting beads (blank calibration particles) during acquisition by flow cytometry, as per the manufacturer’s instructions.

  2. Antibody staining of LN cell suspensions
    1. Transfer up to 200 μl of the cell suspension into each well of a 96 U-bottom well plate.
      Note: If plate holders are not available, the staining can be performed in small Eppendorf tubes and a refrigerated micro-centrifuged can be used. Alternatively, staining can be performed in FACS tubes.
    2. Centrifuge plate at 500 x g for 5 min and at 4 °C then flick off the supernatant.
    3. Add 50 μl of the stromal cell antibody cocktail mix (see Recipes) to each well, resuspend the cells and incubate for 25 min at 4 °C in the dark.
    4. Add 150 μl of FACS buffer per well, centrifuge the plate and flick off the supernatant.
    5. Resuspend cell suspension in 200 μl of FACS buffer and run the stained cells in a flow cytometer (BD LSRFORTESSA).

Data analysis

It is recommended to include 4-5 mice per group and perform 2-3 independent experiments. Flow cytometry files are analysed using FlowJo to gate and quantitate individual stromal cells subsets (Figure 1). To analyse LN stromal cell subsets during infection, draining and non-draining LN are compared, and populations of stromal cells quantitated and numbers plotted (Figure 2). This procedure will also release all leukocytes from the LN, enabling additional antibody staining and analysis of other cell types if required (see Note 1). To assess statistical significance between groups, unpaired t-tests or Mann-Whitney tests should be performed, as appropriate, using Prism (GraphPad).


Figure 1. Gating strategy used to identify LN stromal cell subsets. Dead cells and doublet cells are first excluded. Stromal cells lack CD45.2 and Ter119, markers of hematopoietic cells and erythroid cells, respectively. The use of gp38 and CD31 allows the identification of fibroblastic reticular cells (FRC), lymphatic endothelial cells (LEC) and blood endothelial cells (BEC). The marker PNAD is used to identify the BEC that form the high endothelial venules (HEV) in LN. Bar graphs show the quantification of the stromal cell subsets in the brachial LN (bLN), inguinal LN (iLN) and the popliteal LN (pLN) at steady state.


Figure 2. Analysis of stromal cell subsets in draining brachial LN and non-draining inguinal LN by flow cytometry after Herpes simplex virus (HSV) infection. Mice were infected by scarification on the left flank skin with HSV and LN harvested 7 days post-infection for stromal cell analysis. During an immune challenge, stromal cells expand to accommodate the increase of total cellularity in the LN.

Notes

Dispase treatment can cleave certain cell surface markers. We found that typical markers of stromal cells are not affected by our enzymatic digestion method. We routinely analyse leukocyte subsets in the LN and found that CD8α and CD19 staining is diminished when this protocol is applied. The use of CD8β and B220 as alternative markers is recommended.

Recipes

  1. Supplementum Completum (SC)
    For 1 L solution:
    23.83 g HEPES
    6 g L-glutamine
    2 vials benzylpenicillin
    2 g streptomycin sulphate
    70 μl 2-mercaptoethanol
    1 L RPMI
    Filter, aliquot in 25 ml solution into 50 ml tubes and keep at -20 °C
  2. RP-2 solution
    For 500 ml solution:
    500 ml of RPMI
    10 ml decomplemented fetal bovine serum
    20 ml of SC solution
    Note: Keep at 4 °C, no longer than 2 months.
  3. Digestion mix 1 (Collagenase D: 1 mg/ml, DNase I: 0.1 mg/ml)
    For 20 ml solution:
    20 ml RP-2 solution
    20 mg collagenase D
    2 mg DNase I
    Note: Discard at the end of the experiment.
  4. Digestion mix 2 (Collagenase D: 1 mg/ml, DNase I: 0.1 mg/ml, Dispase II 0.8 mg/ml)
    For 20 ml solution:
    20 ml RP-2 solution
    20 mg collagenase D
    2 mg DNase I
    16 mg dispase II
    Note: Discard at the end of the experiment.
  5. FACS buffer
    For 500 ml solution:
    500 ml of PBS
    2.5 g of BSA
    5 ml 0.5 M EDTA
    Note: Keep at 4 °C, no longer than 3 months.
  6. Lymph node stromal cell antibody cocktail
    For 500 μl solution:
    500 μl FACS buffer
    1 μl gp38-PE (1/500)
    1 μl CD31-PE-Cy7 (1/500)
    10 μl CD45.2-A700 (1/50)
    10 μl Ter119-A700 (1/50)
    2.5 μl PNAd-A488 (1/200)
    1 μl Live/Dead cell marker-NearIR (1/500)
    2.5 μl purified CD16/CD32 (1/200)
    Note: Keep at 4 °C or on ice.

Acknowledgments

This protocol was adapted from our publication (Gregory et al., 2017). This work was supported by the Australian Research Council.

References

  1. Gregory, J. L., Walter, A., Alexandre, Y. O., Hor, J. L., Liu, R., Ma, J. Z., Devi, S., Tokuda, N., Owada, Y., Mackay, L. K., Smyth, G. K., Heath, W. R. and Mueller, S. N. (2017). Infection programs sustained lymphoid stromal cell responses and shapes lymph node remodeling upon secondary challenge. Cell Rep 18(2): 406-418.

简介

我们的方案描述了从淋巴结(LN)分离基质细胞的简单过程。 LN被破坏,然后用胶原酶和分散酶酶消化以产生可以用荧光标记的抗体染色的单细胞悬浮液并通过流式细胞术分析。 该方案能够识别形成LN高内皮小静脉(HEV)的PNAd + BEC的成纤维细胞网状细胞(FRC),淋巴内皮细胞(LEC),血液内皮细胞(BEC)。 该方法可以用于检测由感染或免疫佐剂诱导的炎症事件期间的LN基质细胞反应,并且在LN中发现大多数白细胞。
【背景】淋巴结(LN)由间充质和内皮基质细胞的复杂网络构成。这些包括成纤维细胞网状细胞(FRCs),淋巴内皮细胞(LECs)和血液内皮细胞(BECs)。这些基质细胞组织了LN的复杂微结构,使得能够支持免疫细胞迁移,体内平衡,耐受性和细胞相互作用,以引发对病原体和肿瘤的免疫应答。我们已经表明,LN基质细胞可以响应于炎症信号而增殖和扩张,并且将免疫细胞募集到伴随感染的LN中(Gregory等,2017)。这些基质细胞也可以显着调节其转录程序以应对感染,从而支持持续的免疫应答。该方案使稳定状态和疾病期间LN的基质细胞亚群可靠地分离。这使得LN基质细胞的表型,功能,遗传或表观遗传学研究揭示了它们如何有助于组织体内平衡和免疫应答。

关键字:淋巴基质细胞, 淋巴结, 内皮细胞, 成纤维网状细胞, 免疫反应, 病毒感染

材料和试剂

  1. 1000μl移液器吸头(海王星,目录号:BT1250)
  2. 200μl移液器吸头(海王星,目录号:BT200)
  3. 20μl移液器吸头(海王星,目录号:BT20)
  4. 10μl移液器吸头(海王星,目录号:BT10XLS3)
  5. 24孔板(Corning,目录号:3527)
  6. 10ml管(SARSTEDT,目录号:62.9924.284)
  7. (Greiner Bio One International,目录号:227261)
  8. 尼龙网70μm(Clear Edge,目录号:PA75-1750)
  9. 5ml聚苯乙烯圆底管(Corning,Falcon ®,目录号:352008)
  10. 96孔圆底板(康宁,目录号:3788)
  11. Spatula(VWR,目录号:10806-408)
  12. C57BL \ 6小鼠(JACKSON LABORATORY,目录号:000664)
  13. 空白校准颗粒(BD,BD Biosciences,目录号:556296)
  14. HEPES(Thermo Fisher Scientific,Gibco TM ,目录号:11344033)
  15. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:21051024)
  16. 苄青霉素(CSL,目录号:AUST R 10329)
  17. 硫酸链霉素(Thermo Fisher Scientific,Gibco TM,目录号:11860038)
  18. 2-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  19. RPMI(Thermo Fisher Scientific,Gibco TM ,目录号:11875093)
  20. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10099141)
  21. 胶原酶D(Sigma-Aldrich,目录号:11088882001)
    制造商:Roche Diagnostics,目录号:11088882001。
  22. Dispase II(Sigma-Aldrich,目录号:04942078001)
    制造商:Roche Diagnostics,目录号:04942078001。
  23. DNase I(Sigma-Aldrich,目录号:DN25-1G)
  24. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:10010023)
  25. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7906-500G)
  26. 乙二胺四乙酸二钠盐二水合物(EDTA)(Sigma-Aldrich,目录号:E5134-1KG)
  27. 抗体
    1. gp38-PE,8.1.1(BioLgend,目录号:127408)
    2. CD31-PE-Cy7,390(BioLegend,目录号:102524)
    3. CD45.2-A700,104(BioLegend,目录号:109822)
    4. Ter-119-A700,TER-119(BioLegend,目录号:116220)
    5. HEV-A488,MECA-79(Thermo Fisher Scientific,eBioscience TM ,目录号:53-6036-82)
    6. CD16 / 32纯化的2.4G2(BD,BD Biosciences,目录号:553142)
  28. Live / Dead可固定的近IR细胞染色试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:L10119)
  29. 补充完成(SC)(见配方)
  30. RP-2解决方案(见配方)
  31. 消化组合1(参见食谱)
  32. 消化组合2(参见食谱)
  33. FACS缓冲区(见配方)
  34. 淋巴结基质细胞抗体鸡尾酒(见食谱)

设备

  1. 水浴(VWR,目录号:89501-464)
  2. Dumont No.5镊子(精细科学工具,目录号:11251-10)
  3. Graefe镊子(Roboz手术器械,目录号:RS-5137)
  4. 称重分析秤(Ohaus,目录号:30208454)
  5. 1,000μl移液器(Eppendorf,目录号:3121000120)
  6. 200μl移液器(Eppendorf,目录号:3121000082)
  7. 20μl移液器(Eppendorf,目录号:3121000040)
  8. 10μl移液器(Eppendorf,目录号:3121000015)
  9. 具有板载体的冷冻离心机(Beckman Coulter,型号:Allegra X-15R,目录号:B09822; Beckman Coulter,目录号:368914)
  10. 流式细胞仪(BD,BD Biosciences,型号:BD LSRFORTESSA)

软件

  1. FlowJo(FlowJo)
  2. 棱镜(GraphPad)

程序

  1. 淋巴结的分离和消化
    1. 准备消化混合物1和2(参见食谱)并置于37°C的水浴中。
      注意:制备的消化混合物的体积取决于实验所需的样品数量。对于多达6个皮肤引流淋巴结(LN),需要1ml的每种消化混合物。消化混合物为每个实验准备新鲜。我们为每个实验准备所需量的每种酶。然而,用户可以准备100x的酶(RPMI),并将其储存在-20°C,以便进一步使用。
    2. 从小鼠收获LN,置于每孔含有1ml RP-2溶液的24孔板的孔中(参见食谱)并保持在冰上。
      注意:我们通常从未感染的小鼠(腹股沟,腋窝或肱动脉)处理两次皮肤排水LN。在炎症发作如单纯疱疹病毒(HSV)侧感染期间,我们单独处理肱动脉LN。如果肛门LN被隔离,我们从非感染的小鼠中汇集并处理至少4个LN,并且在炎性事件期间只处理一个LN。
      为了帮助识别小鼠LN,用户可以在鼠标模型中参考LN的本地化图,这里可以使用:
      http://www.informatics .jax.org / greenbook / figures / figure13-4.shtml
    3. 将LN从板中取出,并用两根薄镊子将LN破坏为4-5块。
    4. 将LN转移到10ml管中,加入1ml消化液1 注意:在此步骤中,LN不会放回到24孔板中。因此,它们用消化混合物1消化1ml。
    5. 将管置于水浴中,37℃孵育25分钟。
    6. 在消化过程中倒置管并轻拂数次。
    7. 孵育后切断1毫升移液管的尖端,彻底混合消化的LN 30秒,最长1分钟。保持不受干扰,直到剩余的可见的组织沉淀到管的底部。
    8. 去除上清液而不取出未消化的LN片段,并转移到含有15ml FACS缓冲液的50ml管中(参见食谱)并保持在冰上。
      注意:确保尽可能多地除去上清液,而不会取出LN的任何部分。管中应留下最少的介质。一轮消化不足以正确消化LN,因此需要进行两轮消化以最大限度地提高待释放的SC的产量。
    9. 向步骤A7中剩余的未消化的LN片中加入1ml消化混合物2,并在37℃的水浴中放置15分钟。
    10. 孵育后,用1 ml移液管彻底混合消化的LN 1分钟。
    11. 将上清液转移到含有来自步骤A8的细胞悬液的管中。
      注意:在此步骤后,LN应完全消化。如果有剩余部分,请仅重复步骤A9和A10,只需10分钟。
    12. 离心细胞悬浮液在500 x g,4℃下5分钟。
    13. 吸出上清液,并将细胞悬液悬浮于500(最多6个皮肤排泄LN或po LN)或1,000μlFACS缓冲液(如果超过6次皮肤排泄LN)。
    14. 在5ml FACS管中通过70μm尼龙网过滤细胞悬浮液并计数细胞。
      注意:根据制造商的说明,用户还可以通过流式细胞术在采集期间使用计数珠(空白校准颗粒)。

  2. LN细胞悬液的抗体染色
    1. 将最多200μl的细胞悬浮液转移到96 U底孔板的每个孔中。
      注意:如果不使用平板支架,则可以在小型Eppendorf管中进行染色,也可以使用冷藏式微离心机。或者,可以在FACS管中进行染色。
    2. 离心板在500×g下5分钟,然后在4℃下从上清液中甩出。
    3. 向每个孔中加入50μl基质细胞抗体混合物混合物(参见食谱),重悬细胞,并在黑暗中4℃孵育25分钟。
    4. 每孔加入150μl的FACS缓冲液,离心板并从上清液中甩出。
    5. 将细胞悬浮液悬浮于200μlFACS缓冲液中,并在流式细胞仪(BD LSRFORTESSA)中运行染色的细胞。

数据分析

建议每组包括4-5只小鼠,并进行2-3次独立实验。使用FlowJo分析流式细胞术文件以门和定量个体基质细胞亚群(图1)。在感染期间分析LN基质细胞亚群,比较排水和非排水LN,定量基质细胞群体并绘制数字(图2)。该程序还将从LN释放所有白细胞,如果需要,可以进行额外的抗体染色和其他细胞类型的分析(见注1)。为了评估组之间的统计学显着性,应使用Prism(GraphPad)执行不成对的测试或Mann-Whitney测试。


图1.用于鉴定LN基质细胞亚群的门控策略。首先排除死细胞和双重细胞。基质细胞分别缺乏造血细胞和红细胞标志物的CD45.2和Ter119。使用gp38和CD31允许鉴定成纤维细胞网状细胞(FRC),淋巴内皮细胞(LEC)和血液内皮细胞(BEC)。标记PNAD用于鉴定在LN中形成高内皮小静脉(HEV)的BEC。条形图显示在稳定状态下肱动脉LN(bLN),腹股沟LN(iLN)和al状LN(pLN)中基质细胞亚群的量化。


图2.在单纯疱疹病毒(HSV)感染后通过流式细胞术分析排水臂和非排水腹股沟LN中的基质细胞亚群小鼠在左侧皮肤上用HSV和LN在感染后7天收获用于基质细胞分析。在免疫攻击期间,基质细胞扩大以适应LN中总细胞数量的增加。

笔记

分解酶处理可以切割某些细胞表面标志物。我们发现基质细胞的典型标记物不受我们的酶消化方法的影响。我们通常分析LN中的白细胞亚群,发现当应用该方案时,CD8α和CD19染色减弱。推荐使用CD8β和B220作为替代标记。

食谱

  1. 补充完成(SC)
    对于1L解决方案:
    23.83 g HEPES
    6克L-谷氨酰胺
    2瓶苄青霉素
    2克硫酸链霉素
    70μl2-巯基乙醇
    1 L RPMI
    过滤,分装在25 ml溶液中,置于50ml管中,保持-20°C
  2. RP-2解决方案
    对于500毫升溶液:
    500毫升RPMI
    10 ml补充的胎牛血清
    20 ml SC溶液
    注意:保持在4°C,不超过2个月。
  3. 消化混合物1(胶原酶D:1mg / ml,DNA酶I:0.1mg / ml)
    对于20ml溶液:
    20 ml RP-2溶液
    20毫克胶原酶D
    2mg DNase I
    注意:在实验结束时丢弃。
  4. 消化混合物2(胶原酶D:1mg / ml,DNA酶I:0.1mg / ml,Dispase II 0.8mg / ml)
    对于20ml溶液:
    20 ml RP-2溶液
    20毫克胶原酶D
    2mg DNase I
    16 mg dispase II
    注意:在实验结束时丢弃。
  5. FACS缓冲区
    对于500毫升溶液:
    500毫升PBS
    2.5g BSA
    5 ml 0.5 M EDTA
    注意:保持在4°C,不超过3个月。
  6. 淋巴结基质细胞抗体鸡尾酒
    对于500μl溶液:
    500μlFACS缓冲液
    1μlgp38-PE(1/500)
    1μlCD31-PE-Cy7(1/500)
    10μlCD45.2-A700(1/50)
    10μlTer119-A700(1/50)
    2.5μlPNAd-A488(1/200)
    1μl活/死细胞标记 - 近红(1/500)
    2.5μl纯化的CD16 / CD32(1/200)
    注意:保持在4°C或冰上。

致谢

这个协议是从我们的出版物(Gregory et al。,et al。,2017)改编而成。这项工作得到澳大利亚研究委员会的支持。

参考

  1. 格雷戈里,JL,Walter,A.,Alexandre,YO,Hor,JL,Liu,R.,Ma,JZ,Devi,S.,Tokuda,N.,Owada,Y.,Mackay,LK,Smyth,GK,Heath ,WR和Mueller,SN(2017)。感染计划持续的淋巴样基质细胞反应并形成二次攻击后的淋巴结重塑。细胞代谢(18)(2):406-418。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Alexandre, Y. O. and Mueller, S. N. (2017). Isolation and Analysis of Stromal Cell Populations from Mouse Lymph Nodes. Bio-protocol 7(16): e2445. DOI: 10.21769/BioProtoc.2445.
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