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Whole Spleen Flow Cytometry Assay
完整脾脏的流式细胞分析

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Abstract

In the Whole Spleen Flow Cytometry Assay, we used splenocytes directly ex vivo for stimulation with a variety of TLR ligands. The splenocytes were stimulated for a total of 4 hours, then stained for intracellular cytokines. We then examined cytokine production via flow cytometry. This allowed us to compare the responses of minimally manipulated primary macrophages/monocytes and conventional dendritic cells.

Materials and Reagents

  1. PerCP/Cy5.5 anti-mouse CD11b (Biolegend, catalog number: 101228 )
  2. PE/Cy7 anti-mouse CD11c (BD Bioscience, catalog number: 558079 )
  3. APC/Cy7 anti-mouse CD19 (BD Bioscience, catalog number: 557655 )
  4. APC anti-mouse CD3e (Biolegend, catalog number: 100312 )
  5. FITC anti-mouse TNFa (Caltag, catalog number: RM9011 )
  6. PE anti-mouse IL-12 (BD Bioscience, catalog number: 554479 )
  7. aCD16/32 FcBlock (BD Bioscience, catalog number: 553142 )
  8. FITC anti-mouse CD8 (BD Bioscience, catalog number: 553031 )
  9. PE anti-mouse CD8 (BD Bioscience, catalog number: 553033 )
  10. PerCP/Cy5.5 anti-mouse CD4 (Biolegend, catalog number: 100539 )
  11. PE/Cy7 anti-mouse CD8 (Biolegend, catalog number: 100722 )
  12. APC anti-mouse CD8 (BD Bioscience, catalog number: 553035 )
  13. Ultrapure O111:B4 EC LPS (Life Technologies, InvitrogenTM, catalog number: tlrl-pelps )
  14. Synthetic Lipid IVa (Peptides International, catalog number: CLP-24006-S )
  15. ODN1826 (Coley Pharmaceuticals)
  16. Frosted glass slides, nonsterile (Fisherbrand, catalog number: 12-550-343 )
  17. Cytofix/Cytoperm Solution (BD Biosciences, catalog number: 554722 )
  18. 10x Perm Wash Buffer (BD Biosciences, catalog number: 554723 )
  19. 10% Paraformaldehyde (Electron Microscopy Sciences, catalog number: 15712-S )
  20. RPMI 1640 Medium (with glutamax, HEPES, Phenol red) (Life Technologies, catalog number: 72400-120 )
  21. Fetal bovine serum (FBS) (Heat Inactivated) (Hyclone, catalog number: SH30070.03HI )
  22. Pen/Strep (Life Technologies, catalog number: 15140-122 )
  23. Beta-ME (Sigma-Aldrich, catalog number: M-7522 )
  24. NaCl
  25. KCl
  26. KH2PO4
  27. Na2HPO4
  28. NaOH
  29. NH4Cl
  30. NaHCO3
  31. 10x Phosphate buffered saline (PBS) (see Recipes)
  32. 1x PBSA/azide (PBSA/az) (see Recipes)
  33. RBC Lysis Buffer (see Recipes)
  34. Culture Medium (see Recipes)
  35. Brefeldin A (Sigma-Aldrich, catalog number: B6542 ) (see Recipes)

Equipment

  1. 15 ml conical tubes (BD Biosciences, Falcon®, catalog number: 352196 )
  2. 50 ml conical tubes (BD Biosciences, Falcon®, catalog number: 352070 )
  3. FACS titertube microtubes (Bio-Rad Laboratories, catalog number: 223-9391 )
  4. 70 μM filters (BD Biosciences, Falcon®, catalog number: 352350 )
  5. Tissue culture (TC) dish (100 x 20 mm) (Corning Incorporated, catalog number: 430167 )
  6. 96-well round bottom plates (Corning Incorporated, catalog number: 3799 )
  7. BD FACS Canto I (6 color analyzer) flow cytometer: Equipped with 2 lasers; blue laser 488 nm four color detection and red laser 633 nm 2 color detection. Uses FACSDiva software
  8. Centrifuge (Beckman Coulter, model: Allegra X-15R )

Software

  1. FACSDiva software
  2. BD FACS Canto I software

Procedure


  1. Splenocyte Isolation
    1. Extract whole spleen from mouse and place in a 15 ml polypropylene tube on ice (no liquid).
    2. Put 5 ml RBC lysis buffer into 15 ml tube with spleen.
    3. Pour out spleen with RBC lysis buffer into a 10 cm tissue culture dish.
    4. Smash spleen thoroughly between frosted glass slides by placing the spleen on the rough side of the frosted part of the slide (wetted with RBC lysis buffer) and grind it between the two slides until spleen is dissociated (Video 1).

      Video 1. Homogenization of spleen between two glass slides

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player

    5. Pipette up the RBC lysis containing splenocytes back into the 15 ml tube.
    6. Add 10 ml of culture medium to the tube, invert 2-3 times.
    7. Spin at 1,300 rpm at 4 °C for 5 min.
    8. Discard the supernatant and resuspend the pellet in 3 ml medium.
    9. Filter through a 70 μM filter over a 50 ml conical tube.
    10. Count 1:100 dilution of cells.
    11. Dilute single-cell suspensions to 1 x 107 cells/ml in RPMI medium (for Flow Assays, counting and calculating of cells is not necessary).
    12. Place splenocytes on ice until ready to plate into 96-well plate.

  2. Stimulation of splenocytes
    1. Prepare a stock of RPMI medium with Brefeldin A added to it at 10 μg/ml.
    2. Vortex and spin down all LPS stocks, then sonicate each for 10 min. After sonication, vortex and spin down again.
    3. Each stimulus (EC LPS, PA, YP, Lipid IVa) is prepared to twice (2x) its desired final concentration using the Brefeldin A containing medium.
    4. Plate 100 μl of each stimulus into the appropriate wells of the 96-well plate, include unstimulated wells as a control.
    5. Plate 100 μl of the splenocytes into the appropriate wells (plate an extra set of splenocytes to be used as single stain controls).
    6. Incubate the plate for 4 h at 37 °C.

  3. Immunocytochemistry (ICC) staining
    1. After 4 h, treat wells with 20 μl of 20 mM EDTA.
    2. Mix and incubate at 37 °C for 10 min.
    3. Mix again after incubation and then spin at 1,300 rpm for 5 min.
    4. Aspirate or flick medium off.
    5. Block with 100 μl of diluted FcBlock (1:100 FcBlock in PBSA/az) to all wells.
    6. Incubate on ice for 15 min.
    7. Add 100 μl of PBSA/az, then spin at 1,300 rpm for 5 min.
    8. Aspirate or flick medium off.
    9. Resuspend/stain with 100 μl of Antibody mix: CD11b PerCP-Cy5.5, CD11c PE-Cy7, CD19 APC-Cy7, and CD3 APC (1:100 Ab in PBSA/az) to the stimulated splenocyte samples.
    10. Add 100 μl of PBSA/az to the single stain controls, then add 1 μl of each of these single stain Ab: CD8 FITC, CD8 PE, CD4 PerCP-Cy5.5, CD8 PE-Cy7, CD19 APC-Cy7, and CD8 APC.
    11. Incubate on ice in dark (or wrap in foil) for 20 min.
    12. Add 100 μl PBSA/az, then spin at 1,300 rpm for 5 min.
    13. Aspirate or flick medium off.
    14. Resuspend with 100 μl of BD cytofix/cytoperm solution to all wells.
    15. Incubate on ice in dark for 20 min.
    16. Add 100 μl of 1x BD Perm wash (dilute 10x Perm wash in dH2O) to all wells.
    17. Spin at 1,300 rpm for 5 min, then aspirate or flick medium off.
    18. Resuspend/wash once with 200 μl of 1x BD Perm wash by pipetting up and down a few times to all wells.
    19. Spin at 1,300 rpm for 5 min, then aspirate or flick medium off.
    20. Resuspend/stain with 50 μl of Antibody mix: TNF FITC and IL-12/IL-23p40 PE (1:100 Ab in 1x perm wash) to the stimulated splenocyte samples (add 50 μl of 1x perm wash to the single stain controls).
    21. Incubate on ice in dark for 30 min.
    22. Add 150 μl 1x perm wash to all wells, then spin at 1,300 rpm for 5 min.
    23. Aspirate or flick medium off.
    24. Resuspend/wash once with 200 μl 1x perm wash by pipetting up and down a few times to all wells.
    25. Spin at 1,300 rpm for 5 min, then aspirate or flick medium off.
    26. Resuspend in final volume of 200 μl of 1% Paraformaldehyde solution (dilute 10% Paraformaldehyde in 1x PBS).
    27. Store at 4 °C in dark (or wrapped in foil) until ready for Flow Cytometry (recommended to store no longer than 24 h) – transfer samples from 96-well plate into titer tubes immediately prior to FACS.

  4. Flow Cytometry
    1. On the BD FACS Canto I software, select for a 'New Experiment' to set up.
    2. Select for Area, Height, Width for FSC and SSC; select for Log and Area for the colors FITC, PE, PerCP/Cy5.5, PE/Cy7, APC/Cy7, and APC (under Inspector box).
    3. Create compensation controls and adjust the gating for unstained splenocytes (adjust FSC and SSC voltages as necessary).
    4. Adjust each color of single stains in the voltage panel so that the positive peak is at the 104 mark.
    5. For single stains, use anti-CD4 for PerCP/Cy5.5, anti-CD19 for APC/Cy7, and anti-CD8 for FITC, PE, PE/Cy7, and APC, see Figure 1 for example.


      Figure 1. Example of single stain controls. Splenocytes were stained with the single antibodies listed under IV-5 and voltages adjusted to give depicted histograms.

    6. Record the desired voltages after any adjustments for each of the single stains and unstain, then calculate compensation controls.
    7. Events are now ready to be recorded – set up to collect 500,000 – 1,000,000 events.

Recipes

  1. 10x Phosphate buffered saline (PBS) (for 1 L)
    75 g NaCl
    2 g KCl
    2 g KH2PO4
    11.5 g Na2HPO4
    1 ml 10 N NaOH
    pH to 7.4 and autoclave
  2. 1x PBSA/azide (for 1 L)
    Dilute 10x PBS to 1x PBS using ddH2O
    10 g BSA
    18 ml of 5% NaN3 (final concentration = 0.09% sodium azide)
    Filter sterilize
  3. RBC Lysis buffer
    0.15 M NH4Cl
    1 mM NaHCO3
    0.1 mM EDTA dissolved in sterile irrigation water
    pH to 7.2-7.4 with 1 M HCl
    Filter sterilize
  4. Culture medium
    RPMI 1640 Medium (with glutamax, HEPES, Phenol red)
    10% FBS
    5 ml Pen/Strep
    5 ml 5 mM beta-ME
  5. Brefeldin A
    Received as 25 mg of dry powder
    Dissolve in ethanol to 2 mg/ml and aliquot into dark eppendorf tubes (light sensitive)
    Store at -20 °C

References

  1. Hajjar, A. M., Ernst, R. K., Fortuno, E. S., 3rd, Brasfield, A. S., Yam, C. S., Newlon, L. A., Kollmann, T. R., Miller, S. I. and Wilson, C. B. (2012). Humanized TLR4/MD-2 mice reveal LPS recognition differentially impacts susceptibility to Yersinia pestis and Salmonella enterica. PLoS Pathog 8(10): e1002963.

简介

在全脾流式细胞术测定中,我们直接使用脾细胞离体用各种TLR配体刺激。 将脾细胞刺激总共4小时,然后对细胞内细胞因子染色。 然后我们通过流式细胞术检查细胞因子产生。 这使我们能够比较最小操纵的原代巨噬细胞/单核细胞和常规树突状细胞的反应。

材料和试剂

  1. PerCP/Cy5.5抗小鼠CD11b(Biolegend,目录号:101228)
  2. PE/Cy7抗小鼠CD11c(BD Bioscience,目录号:558079)
  3. APC/Cy7抗小鼠CD19(BD Bioscience,目录号:557655)
  4. APC抗小鼠CD3e(Biolegend,目录号:100312)
  5. FITC抗小鼠TNFα(Caltag,目录号:RM9011)
  6. PE抗小鼠IL-12(BD Bioscience,目录号:554479)
  7. aCD16/32FcBlock(BD Bioscience,目录号:553142)
  8. FITC抗小鼠CD8(BD Bioscience,目录号:553031)
  9. PE抗小鼠CD8(BD Bioscience,目录号:553033)
  10. PerCP/Cy5.5抗小鼠CD4(Biolegend,目录号:100539)
  11. PE/Cy7抗小鼠CD8(Biolegend,目录号:100722)
  12. APC抗小鼠CD8(BD Bioscience,目录号:553035)
  13. Ultrapure O111:B4 EC LPS(Life Technologies,Invitrogen TM,目录号:tlrl-pelps)
  14. 合成脂质IVa(Peptides International,目录号:CLP-24006-S)
  15. ODN1826(Coley Pharmaceuticals)
  16. 磨砂玻璃片,非无菌(Fisherbrand,目录号:12-550-343)
  17. Cytofix/Cytoperm溶液(BD Biosciences,目录号:554722)
  18. 10×Perm洗涤缓冲液(BD Biosciences,目录号:554723)
  19. 10%多聚甲醛(Electron Microscopy Sciences,目录号:15712-S)
  20. RPMI 1640培养基(含glutamax,HEPES,酚红)(Life Technologies,目录号:72400-120)
  21. 胎牛血清(FBS)(热灭活)(Hyclone,目录号:SH30070.03HI)
  22. Pen/Strep(Life Technologies,目录号:15140-122)
  23. β-ME(Sigma-Aldrich,目录号:M-7522)
  24. NaCl
  25. KCl
  26. KH 2 PO 4
  27. Na HPO 4
  28. NaOH
  29. NH 4 Cl
  30. NaHCO 3
  31. 10x磷酸盐缓冲盐水(PBS)(见Recipes)
  32. 1x PBSA /叠氮化物(PBSA/az)(请参阅配方)
  33. RBC裂解缓冲液(参见配方)
  34. 培养基(见配方)
  35. 布雷菲德菌素A(Sigma-Aldrich,目录号:B6542)(参见Recipes)

设备

  1. 15ml锥形管(BD Biosciences,Falcon ,目录号:352196)
  2. 50ml锥形管(BD Biosciences,Falcon ,目录号:352070)
  3. FACS钛管微管(Bio-Rad Laboratories,目录号:223-9391)
  4. 70μM滤器(BD Biosciences,Falcon ,目录号:352350)
  5. 组织培养(TC)皿(100×20mm)(Corning Incorporated,目录号:430167)
  6. 96孔圆底板(Corning Incorporated,目录号:3799)
  7. BD FACS Canto I(6色分析仪)流式细胞仪:配备2个激光器; 蓝色激光488 nm四色检测和红色激光633 nm 2颜色检测。 使用FACSDiva软件
  8. 离心机(Beckman Coulter,型号:Allegra X-15R)

软件

  1. FACSDiva软件
  2. BD FACS Canto I软件

程序


  1. 脾细胞分离
    1. 从小鼠提取整个脾脏,放在15毫升聚丙烯管在冰上(没有液体)
    2. 将5 ml RBC裂解缓冲液加入15ml含有脾的管中
    3. 将脾脏用RBC裂解缓冲液倒入10cm组织培养皿中
    4. 通过将脾脏放置在载玻片的磨砂部分的粗糙侧上(用RBC裂解缓冲液润湿),在磨砂玻璃载玻片之间彻底地脾脏磨碎,并在两个载玻片之间研磨直到脾 (视频1)。

      视频1.两个玻璃片之间的脾匀浆
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    5. 吸取含有脾细胞的RBC裂解液回到15ml管中
    6. 向管中加入10ml培养基,倒转2-3次
    7. 在4℃下以1,300rpm旋转5分钟
    8. 弃去上清液并将沉淀重悬在3ml培养基中。
    9. 通过50μm锥形管过滤通过70μM过滤器
    10. 计数1:100稀释的细胞
    11. 在RPMI培养基中稀释单细胞悬浮液至1×10 7个细胞/ml(对于流动分析,不需要计数和计算细胞)。
    12. 将脾细胞置于冰上,直到准备平板到96孔板。

  2. 刺激脾细胞
    1. 准备加入Brefeldin A的RPMI培养基的储备液,浓度为10μg/ml
    2. 涡旋和旋转所有LPS股票,然后每个声波10分钟。 超声后,再次涡旋和旋转
    3. 使用含布雷菲德菌素A的培养基将每种刺激(EC LPS,PA,YP,脂质IVa)制备成其所需最终浓度的两倍(2x)。
    4. 将100μl的每种刺激平板接种到96孔板的合适孔中,包括未刺激的孔作为对照
    5. 板100微升的脾细胞进入适当的孔(板额外的脾细胞作为单一的污点控制)。
    6. 将板在37℃下孵育4小时。

  3. 免疫细胞化学(ICC)染色
    1. 4小时后,用20μl20mM EDTA处理孔
    2. 混合并在37℃孵育10分钟
    3. 孵育后再次混合,然后以1,300rpm旋转5分钟
    4. 吸出或闪烁介质。
    5. 用100μl稀释的FcBlock(PBSA/az中的1:100FcBlock)封闭到所有孔中
    6. 在冰上孵育15分钟。
    7. 加入100μlPBSA/az,然后以1,300rpm旋转5分钟
    8. 吸出或闪烁介质。
    9. 用刺激的脾细胞样品用100μl抗体混合物:CD11b PerCP-Cy5.5,CD11c PE-Cy7,CD19 APC-Cy7和CD3 APC(PBSA/az中的1:100 Ab)重悬/染色。
    10. 向单染色对照中加入100μlPBSA/az,然后加入1μl每种这些单染色Ab:CD8 FITC,CD8 PE,CD4 PerCP-Cy5.5,CD8 PE-Cy7,CD19 APC-Cy7和CD8 APC。
    11. 在冰上在黑暗中(或包装箔)孵育20分钟
    12. 加入100μlPBSA/az,然后以1,300rpm旋转5分钟
    13. 吸出或闪烁介质。
    14. 用100μlBD cytofix/cytoperm溶液重悬于所有孔中
    15. 在冰上在黑暗中孵育20分钟。
    16. 向所有孔中加入100μl1×BD Perm洗涤(在dH 2 O中稀释10×Perm洗涤)。
    17. 以1,300rpm旋转5分钟,然后吸出或轻弹介质
    18. 通过向所有孔中上下吹打数次,用200μl1x BD Perm洗涤液重悬/洗涤一次。
    19. 以1,300rpm旋转5分钟,然后吸出或轻弹介质
    20. 用50μl抗体混合物:TNF FITC和IL-12/IL-23p40PE(1×Perm洗涤中的1:100 Ab)重悬/染色至刺激的脾细胞样品(向单染色对照中加入50μl1x perm洗涤液) 。
    21. 在冰上在黑暗中孵育30分钟。
    22. 向所有孔中加入150μl1x Perm洗涤液,然后以1,300rpm旋转5分钟
    23. 吸出或闪烁介质。
    24. 用200μl1次Perm洗涤重悬/洗涤一次,上下吹打几次到所有孔
    25. 以1,300rpm旋转5分钟,然后吸出或轻弹介质
    26. 重悬于最终体积为200μl的1%多聚甲醛溶液(稀释10%多聚甲醛的1×PBS溶液)。
    27. 存储在4℃黑暗(或包装在箔),直到准备好流式细胞术(建议存储不超过24小时) - 在FACS之前立即将样品从96孔板转移到滴度管。

  4. 流式细胞术
    1. 在BD FACS Canto I软件上,选择"新实验"进行设置。
    2. 选择面积,高度,宽度的FSC和SSC;选择日志和区域颜色FITC,PE,PerCP/Cy5.5,PE/Cy7,APC/Cy7和APC(在检查器框下)。
    3. 创建补偿控制并调整未染色脾细胞的门控(必要时调整FSC和SSC电压)
    4. 调整电压面板中单个污渍的每种颜色,使正峰位于10 4 标记处。
    5. 对于单染色,对于PerCP/Cy5.5使用抗CD4,对于APC/Cy7使用抗CD19,对于FITC,PE,PE/Cy7和APC使用抗CD8,例如参见图1。

      图1.单染色对照的实例。用IV-5列出的单个抗体染色脾细胞,并调整电压以得到所描绘的直方图。

    6. 在对每个单个污渍和未污染的任何调整后记录所需的电压,然后计算补偿控制
    7. 现在可以记录事件 - 设置为收集500,000 - 1,000,000个事件。

食谱

  1. 10x磷酸盐缓冲盐水(PBS)(1L) 75克NaCl
    2克KCl
    2g KH sub 2 PO 4 sub
    11.5g Na 2 HPO 4
    1ml 10N NaOH
    pH至7.4,高压釜
  2. 1x PBSA /叠氮化物(1 L)
    使用ddH 2 O稀释10x PBS至1x PBS 10 g BSA
    18ml 5%NaN 3(终浓度= 0.09%叠氮化钠) 过滤灭菌
  3. RBC裂解缓冲液
    0.15 M NH 4 Cl
    1mM NaHCO 3/v/v 0.1mM EDTA溶于无菌灌注水中 用1M HCl将pH调节至7.2-7.4 过滤灭菌
  4. 培养基
    RPMI 1640培养基(含glutamax,HEPES,酚红)
    10%FBS
    5ml Pen/Strep
    5ml 5mMβ-ME
  5. 布雷菲德菌素A
    接受25mg干粉
    溶于乙醇至2 mg/ml,并分装入黑色eppendorf管(光敏)
    储存于-20°C

参考文献

  1. Hajjar,A.M.,Ernst,R.K.,Fortuno,E.S.,3rd,Brasfield,A.S.Yay,C.S.,Newlon,L.A.,Kollmann,T.R.,Miller,S.I。和Wilson, 人源化TLR4/MD-2小鼠显示LPS识别差异影响对鼠疫耶尔森氏菌的敏感性< 8(10):e1002963。
  • 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. Yam, C. S. and Hajjar, A. M. (2013). Whole Spleen Flow Cytometry Assay. Bio-protocol 3(15): e834. DOI: 10.21769/BioProtoc.834.
  2. Hajjar, A. M., Ernst, R. K., Fortuno, E. S., 3rd, Brasfield, A. S., Yam, C. S., Newlon, L. A., Kollmann, T. R., Miller, S. I. and Wilson, C. B. (2012). Humanized TLR4/MD-2 mice reveal LPS recognition differentially impacts susceptibility to Yersinia pestis and Salmonella enterica. PLoS Pathog 8(10): e1002963.
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