Transfection of Human Naive CD4+ T Cells with PHA Activation and Neon Electroporation
采用PHA活化和Neon电穿孔法转染人幼稚CD4+T淋巴细胞   

引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
The Journal of Immunology
Mar 2013

Abstract

Transfection of primary T cells can be challenging. This protocol describes a method to transfect primary human naive CD4+ T cells with an AP-1 luciferase reporter using low-level activation by phytohemagglutinin (PHA) and electroporation, as published (Palin et al., 2013). This technique is a modification of one previously described by our group (Cron et al., 2013). Anyone wishing to transfect murine T cells should consult the publication by Cron et al., 2013. This technique may be adapted for other primary T cell types by optimizing the Neon electroporation conditions, as described in the text. Other luciferase or GFP reporters may be used, and will require optimization of the stimulation conditions for that particular reporter.

Keywords: Human CD4+ T cells (人体的CD4 + T细胞), Transfection (转染), Electroporation (电穿孔), Luciferase reporter (荧光素酶报告基因), GFP reporter (GFP报告)

Materials and Reagents

  1. Human blood or peripheral blood mononuclear cells (PBMCs), collected using heparin (1 ml per 60 ml of blood as an anti-coagulant)
  2. Heparin (Sigma-Aldrich, catalog number: H3393 )
  3. Ficoll-Hypaque (GE Life Sciences, catalog number: 17-1440-02 )
  4. Human MACS Naive CD4+ T cell II Kit (Miltenyi Biotec, catalog number: 130-094-131 )
  5. BSA (Thermo Fisher Scientific, catalog number: SH30574 )
  6. PBS (Life Technologies, InvitrogenTM, catalog number: 10010 )
  7. RPMI medium (Life Technologies, InvitrogenTM, catalog number: 11875093 )
  8. Heat-inactivated fetal bovine serum (FBS) (Atlanta Biologicals or other supplier)
  9. Hank's balanced salt solution without calcium or magnesium (HBSS) (Life Technologies, InvitrogenTM, catalog number: 14170161 )
  10. PHA (Sigma-Aldrich, catalog number: 61764 )
  11. Neon Transfection System 100 μl kit (Life Technologies, InvitrogenTM, catalog number: MPK10096 or MPK10025 )
  12. Aqua Amine Live/Dead discriminator (Life Technologies, InvitrogenTM, catalog number: L34957 )
  13. 7AAD (BD Biosciences, catalog number: 559925 )
  14. Anti-CD3+ anti-CD28- coated Dynal beads (Life Technologies, InvitrogenTM, catalog number: 11131D )
  15. Ionomycin (Sigma-Aldrich, catalog number: I3909 )
  16. Phorbol myristate acetate (PMA) (Sigma-Aldrich, catalog number: P8139 )
  17. One Glo luciferase reagent (Promega Corporation, catalog numbers: E6110 , E6120 , or E6130 )
  18. AP-1-luciferase reporter (as described in Vaysberg et al., 2008)
    Note: This plasmid consists of a pGL3 backbone (Promega Corporation, catalog number: E1751) with 5 copies of the AP-1 binding site from the metallothionein promoter inserted into the human IL2 minimal promoter.
  19. pEGFP-N1 (Clonetech) or equivalent GFP-expressing plasmid
  20. Beta-actin (b-actin)-driven luciferase reporter plasmid, or other highly expressing luciferase reporter
    Note: Use of a renilla luciferase-expressing plasmid is not recommended in this assay, as we found it interferes with the firefly luciferase signal.
  21. CD3-Alexa700 (eBiosceince, catalog number: 56-0037-42 )
  22. CD4-PE-Cy7 (BD Biosciences, catalog number: 557852 )
  23. CD4-PE (Life Technologies, InvitrogenTM, catalog number: MHCD0404 )
  24. CD45RA–PacificBlue (Life Technologies, InvitrogenTM, catalog number: MHCD45RA28 )
  25. CD45RO-PerCp-Cy5.5 (BD Biosciences, catalog number: 560607 )
  26. CD4RA-APC (Life Technologies, InvitrogenTM, catalog number: MHCD45RA05 ) or Alexa647 (BD Biosciences, catalog number: 562763 )
  27. CD25-APC (Life Technologies, InvitrogenTM, catalog number: MHCD2505 )
  28. CD40L-FITC (BD Biosciences, catalog number: 555699 )
  29. CD62L-PE (BD Biosciences, catalog number: 555544 )
  30. CD69-PE-Cy5 (Life Technologies, InvitrogenTM, catalog number: MHCD6906 )
  31. MACS buffer (see Recipes)

Equipment

  1. 96-well round-bottom sterile tissue culture plates (BD Biosciences, catalog number: 353077 or equivalent)
  2. 96-well white-wall, flat-bottom plates (BD Biosciences, catalog number: 353296 or equivalent)
  3. MACS LS columns (Miltenyi Biotec, catalog number: 130-042-401 )
  4. Neon Transfection System (Life Technologies, InvitrogenTM, model: MPK5000 )
  5. Dynal magnet (Life Technologies, InvitrogenTM, model: 12321D )
  6. Luminometer with 96-well plate capability (any manufacturer)
  7. MACS midi magnets (Miltenyi Biotec, model: 130-042-302 )
  8. Flow cytometer (BD LSR II or similar)

Procedure

Note: All centrifugation steps in 15 or 50 ml conical tubes should be performed at 450 x g for 10 minutes in a general-purpose centrifuge. All centrifugation steps in eppendorf tubes should be performed at 800 x g for 5 min in a microfuge.

  1. Subject blood to standard Ficoll-Hypaque density gradient centrifugation; harvest interface layer and wash twice in HBSS, and purify with MACS Hunan Naive CD4+ II Kit and LS columns. Collect the flow-through; this is the naive CD4+ T cell fraction. Do not add EDTA to MACS buffer; use only 0.5% BSA in sterile PBS. Count cells. Resuspend in RPMI and centrifuge 540 x g for 10 minutes in general purpose centrifuge. You will need at least 1 x 106 cells for each plasmid to be transfected. To allow for extra volume, stimulate a minimum 1.2 x 107 cells. See Table 1 for a breakdown of cell number requirements.

    Table 1.  Plasmids and number of replicates
    Plasmid
    Number of Replicates
    AP-1 luciferase reporter
    5
    b-actin luciferase reporter
    1
    pEGFP reporter
    1
    No plasmid (mock)
    3

  2. Dilute PHA to 2.5 μg/ml in RPMI/10% FBS (no PSG). Concentration should be optimized for each PHA source in terms of transfection efficiency and low expression of activation markers. Resuspend cells in PHA/RPMI at 1 x 106 cells/100 μl. Leave 1 x 106 cells unstimulated for FACS analysis of activation markers.
  3. Activate cells for 19.5-20 h at 37 °C in a humidified 5% CO2 containing incubator. Set concurrent timers for 19.5 and 20 h. If you are doing more than 10 transfections, stagger the start of the stimulations by at least half an hour to allow enough time to finish transfections by 20 h. The samples must be transfected between 19.5 and 20 h after PHA stimulation.
  4. Before 19.5 h is up, add DNA plasmids to Eppendorf tubes (2.0 μg/1 x 106 cells), as shown in Table 2. Aliquot 1.0 ml RPMI/10% FBS (no antibiotics) to wells of a 24 well plate (one well for each transfection reaction), and keep at 37 °C until ready to transfect. Add 3.0 ml Neon buffer E (included in Neon kit) to transfection chamber.

    Table 2. Volumes for transfections
    Transfection
    DNA
    Final volume
    Total number of
    reactions

    buffer T
    cells necessary
    1
    3.0 μg
    150 μl
    1.5 x 106
    2
    5.0 μg
    250 μl
    2.5 x 106
    3
    7.0 μg
    350 μl
    3.5 x 106
    n
    2 n + 1 μg
    100 n + 50 μl
    n x 106 + 5 x 105

  5. At 19.5 h, begin washing cells in PBS by centrifuging for 800 x g for 5 min in microfuge. Resuspend cells in Neon buffer T (106 cells/100 μl, see Table 2). Prepare at least 50 μl extra volume for each tube. Not allowing extra volume will introduce air bubbles into the Neon pipette, which will disrupt the transfection.
  6. Make sure to finish transfections by 20 h. Set the Neon for 2,400 V, 2 pulses, 12 ms. For other human T cell subsets, these parameters should be optimized as described in the Neon manual.
  7. Electroporate 100 μl at a time. Check tip to make sure there are no bubbles in the Neon tip, and watch for sparks. If a sample sparks, omit it and do not reuse that tip. Use tips no more than twice. Add each electroporation/transfection reaction (100 μl) to 1 well of 24 well plate. Repeat electroporation until finished. Work quickly to maintain consistency. To assess viability, you may electroporate one sample in the absence of plasmid.
  8. Incubate cells for 24 h in 37 °C/5% CO2 incubator.
  9. After 24 or 48 h of activation, stain cells for activation markers. Stain one unstimulated sample and one PHA-stimulated sample. Include aqua amine live/dead discriminator. To stain, pellet 1 x 106 cells in an eppendorf tube (800 x g, 5 min in microfuge), wash with PBS and follow manufacturer's instructions for aqua amine staining. Wash cells in MACS buffer and resuspend in a total volume of 100 μl MACS buffer with antibodies as listed below. Incubate for 10 minutes at room temperature, wash with 1.0 ml of MACS buffer, and resuspend in 1% paraformaldehyde in MACS buffer. Suggested antibodies and fluorophores are listed in the Table 3. This staining combination may be adapted according to the capability of the available flow cytometer.

    Table 3. Suggested staining for activation markers
    Epitope
    Fluorophore
    Volume/106 cells
    CD3
    Alexa700
    2.5 μl
    CD4
    PE-Cy7
    10 μl
    CD45RA
    PacificBlue
    2.5 μl
    CD45RO
    PerCp-Cy5.5
    10 μl
    CD25
    APC
    2.5 μ
    CD69
    PE-Cy5
    2.5 μl
    CD40L
    FITC
    10 μl
    CD62L
    PE
    10 μl

    Gate on lymphocytes, aqua amine- cells, singlets. To assess purity, gate on CD3+ CD4+ then CD45RA+ CD45RO- cells. To identify the percentage of cells that are not activated in the PHA-stimulated sample, use the unstimulated sample to set gates using a histogram plot for CD25-, CD69-, CD40L-, and CD62L+. See Figure 1 for gating strategy.


    Figure 1. Analysis of sample purity and activation by flow cytometry. Top row: unstimulated sample stained for purity. Bottom row: Overlays of unstimulated (gray) and PHA-stimulated samples (black) stained for activation markers as indicated, and gated on naive CD4+ T cells as indicated.

  10. At 24 h after transfection, activate cells for measurement of AP-1 activity. Wash anti-CD3+anti-CD28-coated Dynal beads with 1 ml RPMI/10% FBS. Leave tube containing beads on magnet for 1 minute before aspirating medium, while leaving tube containing beads on magnet. Remove from magnet, add 1 ml medium, and repeat for a total of 3 washes. Prepare 25 μl beads per 106 cells, plus enough for 1 extra sample. Resuspend in 4x original volume (e.g. 100 μl RPMI/10% FBS per 25 μl beads originally used).
  11. Dilute and combine PMA and ionomycin to working concentrations of 50 ng/ml PMA and 250 nM ionomycin. Make a total volume of 1 ml.
  12. Transfer transfected cells to eppendorf tubes and pellet (800 x g, 5 min in microfuge). Leave one replicate of unstimulated cells. Do not combine replicates. Aspirate supernatants and resuspend in 550 μl RPMI/10% FBS. Aliquot 100 μl per well and transfer to 96 well round-bottom plate. Split transfection replicates across rows for 5 wells each replicate. Pellet cells by centrifuging at 450 x g for 10 min and aspirate supernatants.
    Note: To avoid this step, you may resuspend cells in 225 μl RPMI/10% FBS and prepare 2x working stocks of all stimulation reagents described above. In this case, aliquot 50 μl of cells per well according to the diagram below and add 50 μl of medium containing stimulation reagents.
  13. Resuspend each well in 100 μl medium containing appropriate stimulation agents and transfer to a 96 well white-wall plate on ice. See Table 4 for layout and diagram of distribution of replicates.
    Use RPMI/10% FBS for unstimulated samples, and for b-actin-luciferase-transfected sample. There is room for additional stimuli, such as anti-CD3 alone. If using soluble antibodies, use a secondary cross-linking antibody.

    Table 4. Sample plate layout for stimulation



    1
    2
    3
    4
    5
    6  
    7  
    8  
    9  
    10
    11
    12
    A
    AP-1 (1)
    medium
    CD3+CD28 Dynal beads
    Iono. + PMA









    B
    AP-1 (2)
    medium
    CD3+CD28 Dynal beads
    Iono. + PMA









    C
    AP-1 (3)
    medium
    CD3+CD28 Dynal beads
    Iono. + PMA









    D
    AP-1 (4)
    medium
    CD3+CD28 Dynal beads
    Iono. + PMA









    E
    AP-1 (5)
    medium
    CD3+CD28 Dynal beads
    Iono. + PMA









    F
    Untransfected
    medium
    medium
    medium
    medium
    medium







    G
    b-actin-luc
    medium
    medium
    medium
    medium
    medium







    H














  14. Once all stimulation reagents have been added, remove plate from ice and incubate for 4 h at 37 °C, 5% CO2.
  15. During stimulation incubation, assess transfection efficiency by staining one untransfected sample and the pEGFP-transfected sample with 2.5 μl of anti-CD4-PE and 2.5 μl of anti-CD4RA-APC or Alexa-647. After staining, add 5 μl 7AAD to each sample to assess viability. This is detected on the PE-Cy5 channel. Do not fix these samples. Transfection efficiency is expected to range between 5 and 40% of live (7AAD-), CD4+ CD45RA+ cells. Set EGFP+ gate at top 1% of untransfected cells. See Figure 2.


    Figure 2. Assessment of viability and transfection efficiency by flow cytometry. Top: mock-transfected sample gated on live cells (7AAD-), then lymphocytes, and CD4+ CD45RA+ cells. Bottom: Measurement of transfection efficiency by %EGFP+ in population described in top row. EGFP+ gate is set at top 1% of signal from mock-transfected cells.

  16. Prepare Promega One-Glo reagent according to the manufacturer's instructions, or thaw an aliquot covered in foil. You will need 100 μl per well. At 4 h after stimulation, remove plate from incubator and add 100 μl One-Glo reagent per well using a multi-channel pipette. Cover with foil and incubate for 3 min.
    Note: There is no need to remove Dynal beads as these do not affect the luciferase reading (A. Palin, unpublished data). Read on a luminometer with a 1 sec read per well. One Glo reagent does not require the use of injectors. Include an empty well as a control for background from the plate.
  17. To analyze data, subtract absorbance values for unstimulated samples, pairing the transfection replicates (i.e. subtract A1 value from A2 or A3; B1 from B2 or B3, etc.). Divide instead of subtract to calculate fold change. The untransfected control gives background fluorescence from the cells and the b-actin luciferase signal is a control for luciferase activity. This will be significantly higher than the stimulated cells. The anti-CD3+CD28- stimulated cells give a measure of the upregulation of AP-1 in response to TCR engagement and co-stimulation, while the ionomycin + PMA serves as a measure of the capacity of the cell to induce AP-1. To compare between two sample types or two groups, use the average of the 5 stimulated replicates minus the unstimulated replicates and an unpaired student's t-test.

Recipes

  1. MACS buffer
    1x PBS
    0.5% BSA
    Sterilize by vacuum filtration.
    Note: Do not include EDTA in this buffer, as it may interfere with T cell receptor signaling.

References

  1. Cron, R. Q., Schubert, L. A., Lewis, D. B. and Hughes, C. C. (1997). Consistent transient transfection of DNA into non-transformed human and murine T-lymphocytes. J Immunol Methods 205(2): 145-150.
  2. Palin, A. C., Ramachandran, V., Acharya, S. and Lewis, D. B. (2013). Human neonatal naive CD4+ T cells have enhanced activation-dependent signaling regulated by the microRNA miR-181a. J Immunol 190(6): 2682-2691.
  3. Vaysberg, M., Hatton, O., Lambert, S. L., Snow, A. L., Wong, B., Krams, S. M. and Martinez, O. M. (2008). Tumor-derived variants of Epstein-Barr virus latent membrane protein 1 induce sustained Erk activation and c-Fos. J Biol Chem 283(52): 36573-36585.

简介

原代T细胞的转染可能是具有挑战性的。 该方案描述了使用植物凝集素(PHA)和电穿孔的低水平激活,用AP-1荧光素酶报道子转染初级人初始CD4 + T细胞的方法,如公开的(Palin等人 。,2013)。 这种技术是先前由我们组描述的技术的修改(Cron等人,2013)。 任何希望转染鼠T细胞的人都应参考Cron等人2013年的出版物。该技术可以通过优化氖电穿孔条件来适应其它原代T细胞类型,如文中所述。 可以使用其他荧光素酶或GFP报道分子,并且将需要优化该特定报道分子的刺激条件。

关键字:人体的CD4 + T细胞, 转染, 电穿孔, 荧光素酶报告基因, GFP报告

材料和试剂

  1. 使用肝素(每60ml血液1ml作为抗凝剂)收集人血或外周血单核细胞(PBMC),
  2. 肝素(Sigma-Aldrich,目录号:H3393)
  3. Ficoll-Hypaque(GE Life Sciences,目录号:17-1440-02)
  4. 人MACS原始CD4 + T细胞II试剂盒(Miltenyi Biotec,目录号:130-094-131)
  5. BSA(Thermo Fisher Scientific,目录号:SH30574)
  6. PBS(Life Technologies,Invitrogen TM ,目录号:10010)
  7. RPMI培养基(Life Technologies,Invitrogen TM,目录号:11875093)
  8. 热灭活的胎牛血清(FBS)(Atlanta Biologicals或其他供应商)
  9. Hank's没有钙或镁的平衡盐溶液(HBSS)(Life Technologies,Invitrogen TM,目录号:14170161)
  10. PHA(Sigma-Aldrich,目录号:61764)
  11. 氖转染系统100μl试剂盒(Life Technologies,Invitrogen TM ,目录号:MPK10096或MPK10025)
  12. Aqua Amine Live/Dead discriminator(Life Technologies,Invitrogen TM ,目录号:L34957)
  13. 7AAD(BD Biosciences,目录号:559925)
  14. 抗-CD3抗体+抗-CD28-涂覆的Dynal珠(Life Technologies,Invitrogen TM,目录号:11131D)
  15. 离子霉素(Sigma-Aldrich,目录号:I3909)
  16. 佛波醇肉豆蔻酸乙酯(PMA)(Sigma-Aldrich,目录号:P8139)
  17. One Glo荧光素酶试剂(Promega Corporation,目录号:E6110,E6120或E6130)
  18. AP-1-荧光素酶报告物(如Vaysberg等人在2008年所述)
    注意:该质粒由具有来自插入人IL2最小启动子的金属硫蛋白启动子的5个拷贝的AP-1结合位点的pGL3骨架(Promega公司,目录号:E1751)组成。
  19. pEGFP-N1(Clonetech)或等价的GFP表达质粒
  20. β-肌动蛋白(b-肌动蛋白) - 驱动的荧光素酶报告质粒或其他高度表达的荧光素酶报告物
    注意:在本试验中不建议使用表达renilla萤光素酶的质粒,因为我们发现它会干扰萤火虫萤光素酶信号。
  21. CD3-Alexa700(eBiosceince,目录号:56-0037-42)
  22. CD4-PE-Cy7(BD Biosciences,目录号:557852)
  23. CD4-PE(Life Technologies,Invitrogen TM,目录号:MHCD0404)
  24. CD45RA-PacificBlue(Life Technologies,Invitrogen TM,目录号:MHCD45RA28)
  25. CD45RO-PerCp-Cy5.5(BD Biosciences,目录号:560607)
  26. CD4RA-APC(Life Technologies,Invitrogen ,目录号:MHCD45RA05)或Alexa647(BD Biosciences,目录号:562763)
  27. CD25-APC(Life Technologies,Invitrogen TM,目录号:MHCD2505)
  28. CD40L-FITC(BD Biosciences,目录号:555699)
  29. CD62L-PE(BD Biosciences,目录号:555544)
  30. CD69-PE-Cy5(Life Technologies,Invitrogen TM,目录号:MHCD6906)
  31. MACS缓冲区(参见配方)

设备

  1. 96孔圆底无菌组织培养板(BD Biosciences,目录号:353077或等同物)
  2. 96孔白壁,平底板(BD Biosciences,目录号:353296或等价物)
  3. MACS LS柱(Miltenyi Biotec,目录号:130-042-401)
  4. 氖转染系统(Life Technologies,Invitrogen TM ,型号:MPK5000)
  5. Dynal磁铁(Life Technologies,Invitrogen TM ,型号:12321D)
  6. 具有96孔板能力的发光计(任何制造商)
  7. MACS中磁体(Miltenyi Biotec,型号:130-042-302)
  8. 流式细胞仪(BD LSR II或类似物)

程序

注意:在15或50ml锥形管中的所有离心步骤应在通用离心机中以450×g离心10分钟。在eppendorf管中的所有离心步骤应在800×g下在微量离心机中进行5分钟。

  1. 将受试者血液标准化至Ficoll-Hypaque密度梯度离心;收获界面层并在HBSS中洗涤两次,并用MACS Hunan原始CD4 +和II柱纯化。收集流通;这是初始CD4 + T细胞部分。不要向MACS缓冲液中加入EDTA;只使用0.5%BSA的无菌PBS。计数单元格。在通用离心机中重悬于RPMI中并在540×g离心10分钟。对于每种待转染的质粒,需要至少1×10 6个细胞。为了允许额外的体积,刺激最少1.2×10 7个细胞。有关细胞数量要求的细目,请参见表1
    表1. 质粒和重复数
    质粒
    复制数量
    AP-1荧光素酶报告物
    5
    b-肌动蛋白荧光素酶报告物
    1
    pEGFP报告物
    1
    无质粒(模拟)
    3

  2. 在RPMI/10%FBS(无PSG)中将PHA稀释至2.5μg/ml。就转染效率和活化标记物的低表达而言,应当针对每种PHA来源优化浓度。在PHA/RPMI中以1×10 6个细胞/100μl重悬细胞。离开1×10 6个未激活用于活化标记物的FACS分析的细胞
  3. 激活细胞19.5-20 h在37℃下,在潮湿的5%CO 2含培养箱。设置并发计时器为19.5和20小时。如果你做超过10次转染,错开刺激的开始至少半小时,以允许足够的时间完成转染20小时。样品必须在PHA刺激后19.5和20小时之间转染
  4. 在19.5小时前,将DNA质粒加入到Eppendorf管(2.0μg/1×10 6个细胞)中,如表2所示。将1.0ml RPMI/10%FBS(无抗生素)的24孔板(每个转染反应一个孔),并保持在37℃直到准备转染。向转染室中加入3.0ml Neon缓冲液E(包含在Neon试剂盒中)
    表2.用于转染的体积
    转染
    DNA
    最终数量
    总数
    反应

    缓冲区T
    必要的单元
    1
    3.0微克
    150μl
    1.5 x 10 6
    2
    5.0微克
    250微升
    2.5 x 10 6
    3
    7.0μg
    350微升
    3.5 x 10 6
    n
    2 n + 1μg
    100 n + 50微升
    n×10 6 + 5×10 5

  5. 在19.5小时,通过在微量离心机中离心800×g离心5分钟开始在PBS中洗涤细胞。在氖缓冲液T中重悬细胞(10 6个细胞/100μl,参见表2)。为每个管准备至少50微升额外的体积。不允许额外的体积将引入气泡进入Neon移液管,这将破坏转染。
  6. 确保完成转染20小时。将氖灯设置为2,400 V,2个脉冲,12 ms。对于其他人类T细胞亚群,这些参数应根据氖手册中的描述进行优化
  7. 每次电穿孔100μl。检查提示,确保霓虹灯提示中没有气泡,并观察火花。如果样品出现火花,请忽略它,不要重复使用该尖端。使用提示不要超过两次。将每个电穿孔/转染反应(100μl)加入24孔板的1孔中。重复电穿孔直到完成。快速工作以保持一致性。为了评估生存力,您可以在没有质粒的情况下电穿孔一个样品
  8. 在37℃/5%CO 2培养箱中孵育细胞24小时
  9. 24或48小时的激活后,染色细胞的激活标记。染色一个未刺激的样品和一个PHA刺激的样品。包括aqua胺活/死区分器。为了染色,在eppendorf管(800×g,在微量离心管中5分钟)中沉淀1×10 6个细胞,用PBS洗涤并遵循制造商的水胺染色说明书。在MACS缓冲液中洗涤细胞,并在总体积为100μl的MACS缓冲液中重悬,其中抗体如下所列。在室温下孵育10分钟,用1.0ml MACS缓冲液洗涤,并重悬于1%多聚甲醛的MACS缓冲液中。建议的抗体和荧光团列于表3中。该染色组合可以根据可用的流式细胞仪的能力进行调整。

    表3.针对激活标记的建议染色
    表位
    荧光团
    Volume/10 6 个单元格
    CD3
    Alexa700
    2.5μl
    CD4
    PE-Cy7
    10微升
    CD45RA
    太平洋
    2.5μl
    CD45RO
    PerCp-Cy5.5
    10微升
    CD25
    APC
    2.5μ
    CD69
    PE-Cy5
    2.5μl
    CD40L
    FITC
    10微升
    CD62L
    PE
    10微升

    门在淋巴细胞,aqua胺细胞,单子。为了评估纯度,在CD3阳性+ CD4 +细胞+ CD45RA阳性细胞和CD45RO阳性细胞上进行门控。为了鉴定在PHA刺激的样品中未被激活的细胞的百分比,使用未刺激的样品使用CD25 - ,CD69L,CD40L - 和CD62L + 。参见图1的门控策略。


    图1.通过流式细胞术分析样品纯度和活化。顶行:未刺激的样品为纯度染色。底部行:如所示的未激活的(灰色)和PHA刺激的样品(黑色)的重叠染色,并且如所示的在初始CD4 + sup/T细胞上门控。

  10. 在转染后24小时,激活细胞用于测量AP-1活性。用1ml RPMI/10%FBS洗涤抗CD3 +抗CD28涂层的Dynal珠。离心管磁珠上的磁珠1分钟,然后吸取培养基,同时留下磁珠上的磁珠。从磁铁中取出,加入1毫升培养基,重复总共3次洗涤。准备25微升每10 6个细胞,加上足够1额外的样品。以4x原始体积(例如100μlRPMI/10%FBS /25μl最初使用的珠子)重悬。
  11. 稀释并结合PMA和ionomycin的工作浓度为50 ng/ml PMA和250 nM离子霉素。使总体积为1 ml。
  12. 将转染的细胞转移到eppendorf管和沉淀(800×g,在微量离心机中5分钟)。留下一个未刺激细胞的复制品。不要合并重复。吸出上清液和重悬在550μlRPMI/10%FBS。等分100微升/孔,并转移到96孔圆底板。分裂转染在每行重复5个孔。通过在450×g离心10分钟并抽吸沉淀细胞 上清液。
    注意:为了避免这个步骤,你可以重悬细胞在225微升RPMI/10%FBS和准备上述所有刺激试剂的2x工作库存。在这种情况下,根据下图,每孔等分50微升细胞,并添加50微升含有刺激试剂的培养基。
  13. 将每个孔重悬于含有适当刺激剂的100μl培养基中,并转移至冰上的96孔白壁板。参见表4的重复分布的布局和分布图 对于未刺激的样品使用RPMI/10%FBS,对于b-肌动蛋白 - 荧光素酶转染的样品。存在额外刺激的空间,例如单独的抗CD3。如果使用可溶性抗体,请使用二级交联抗体
    表4.刺激样品板布局


    1
    2
    3
    4
    5
    6  
    7  
    8  
    9  
    10
    11
    12
    A
    AP-1(1)
    中等
    CD3 + CD28 Dynal珠子
    Iono。 + PMA









    B
    AP-1(2)
    中等
    CD3 + CD28 Dynal珠子
    Iono。 + PMA









    C
    AP-1(3)
    中等
    CD3 + CD28 Dynal珠子
    Iono。 + PMA









    D
    AP-1(4)
    中等
    CD3 + CD28 Dynal珠子
    Iono。 + PMA









    E
    AP-1(5)
    中等
    CD3 + CD28 Dynal珠子
    Iono。 + PMA









    F
    未转播
    中等
    中等
    中等
    中等
    中等







    G
    b-actin-luc
    中等
    中等
    中等
    中等
    中等







    H














  14. 一旦添加了所有刺激试剂,从冰中取出板并在37℃,5%CO 2下孵育4小时。
  15. 在刺激孵育期间,通过染色一个未转染的样品和pEGFP转染的样品用2.5μl抗CD4-PE和2.5μl抗CD4RA-APC或Alexa-647来评估转染效率。 染色后,向每个样品中加入5μl7AAD以评估存活力。 这在PE-Cy5通道上检测。 不要修复这些样品。 预期转染效率范围为活的(7AAD ),CD4 + CD45RA 的5%至40% + 细胞。在未转染细胞的顶部1%处设置EGFP + 门。见图2.


    图2.通过流式细胞术评估存活力和转染效率。上部:在活细胞(7AAD - )上门控的模拟转染的样品,然后淋巴细胞和CD4 + CD45RA + 细胞。底部:在顶行中描述的群体中通过%EGFP + 测量转染效率。 EGFP + Gate设置在来自模拟转染细胞的信号的顶部1%
  16. 根据制造商的说明准备Promega One-Glo试剂,或解冻覆盖在箔中的等分试样。您将需要每孔100μl。刺激后4小时,从培养箱中取出板,并使用多通道移液器添加100微升One-Glo试剂每孔。盖上箔,孵育3分钟。
    注意:没有必要删除Dynal珠,因为这些不影响萤光素酶读数(A.Palin,未发表的数据)。在光度计上读取,每孔读取1秒。 One Glo试剂不需要使用注射器。包括一个空井作为从板的背景的控制。
  17. 为了分析数据,减去未刺激样品的吸光度值,将转染复制品配对(即,从A2或A3中减去A1值;从B2或B3中取出B1,等等。)。除以减法计算倍数变化。未转染的对照产生来自细胞的背景荧光,b-肌动蛋白荧光素酶信号是荧光素酶活性的对照。这将显着高于受刺激的细胞。抗CD3抗体+ CD28抗体刺激的细胞提供AP-1响应于TCR接合和共刺激的上调的测量,而离子霉素+ PMA用作测量细胞诱导AP-1的能力。要比较两种样品类型或两组,使用5个刺激复制品的平均值减去未刺激的复制品和非配对的学生t检验。

食谱

  1. MACS缓冲区
    1x PBS
    0.5%BSA
    通过真空过滤灭菌。
    注意:不要在此缓冲液中加入EDTA,因为它可能会干扰T细胞受体信号传导。

参考文献

  1. Cron,R.Q.,Schubert,L.A.,Lewis,D.B.and Hughes,C.C。(1997)。 将DNA连续瞬时转染到非转化的人和鼠T- 淋巴细胞。 J Immunol Methods 205(2):145-150
  2. Palin,A.C.,Ramachandran,V.,Acharya,S.and Lewis,D.B。(2013)。 人类新生儿幼稚CD4 + T细胞具有增强的由微小RNA miR-181a调节的激活依赖性信号传导。/a> J Immunol 190(6):2682-2691
  3. Vaysberg,M.,Hatton,O.,Lambert,S.L.,Snow,A.L.,Wong,B.,Krams,S.M.and Martinez,O.M。(2008)。 爱泼斯坦 - 巴尔病毒潜伏膜蛋白1的肿瘤衍生变体诱导持续的Erk激活和c-Fos 。 J Biol Chem 283(52):36573-36585。
  • 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。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Palin, A. and Lewis, D. B. (2013). Transfection of Human Naive CD4+ T Cells with PHA Activation and Neon Electroporation. Bio-protocol 3(15): e836. DOI: 10.21769/BioProtoc.836.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。