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In vitro Assays for the Detection of Calreticulin Exposure, ATP and HMGB1 Release upon Cell Death
采用体外分析法检测细胞死亡后的钙网蛋白暴露、ATP和HMGB1释放   

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Abstract

Accumulating evidence is revealing the essential role of immune system in cancer treatment. Certain chemotherapeutic drugs can potently induce the release of ‘cell death associated molecular patterns’ (CDAMPs), which accompanies cancer cell demise. CDAMPs can engage corresponding receptors on immune cells and stimulate immune responses to achieve long-term tumor control (Ma et al., 2013; Ma et al., 2014; Yang et al., 2015). Among reported CDAMPs, calreticulin (CALR), ATP and HMGB1 are well known for their immune-stimulatory effect. Here we describe the assays that we applied to measure cell death and these CDAMPs. Briefly, cell death can be analyzed by co-staining of 4’,6-diamidino-2-phenylindole (DAPI) with 3,3’-Dihexyloxacarbocyanine Iodide [DiOC6(3)] or Annexin V. CALR exposure on the cell membrane can be detected by flow cytometry. ATP and HMGB1 release can be quantified by luminescence assay and ELISA assay respectively.

Keywords: Cell death(细胞死亡), DiOC6(3)(DiOC6(3)), Annexin V(膜联蛋白V), Calreticulin(钙网蛋白), HMGB1(HMGB1), ATP(ATP), Luciferase(荧光素酶)

Background

Lactate dehydrogenase assay and trypan blue staining are traditional methods to examine cell death. We describe here two feasible and economic solutions to detect apoptotic and necrotic cell death by flow cytometry (FCM). DAPI labels cells with disrupted integrity (necrosis), while Annexin V binds to phosphatidylserine (which is externalized upon apoptosis). DiOC6(3) uptake indicates mitochondrial transmembrane potential (MTP) and the collapse of MTP reveals apoptosis. DAPI doesn’t require compensation with phycoerythrin (PE, which is conjugated with Annexin V protein) or DiOC6(3), and therefore show advantage over propidium iodide (PI) in these assays.
   CALR is an endoplasmic reticulum protein, and activation of caspase cascades triggers CALR translocation to cytoplasmic membrane. CALR exposure can be detected by immunofluorescent staining with corresponding antibodies, followed by FCM- or microscopy-based detection. Extracellular ATP can be measured by enzymatic activity of Luciferase, while HMGB1 concentration can be detected by sensitive ELISA kits.

Materials and Reagents

  1. 24 well plates
  2. 2 ml Eppendorf tubes
  3. MCA205 fibrosarcoma cells (H2b) were induced by 3-methylcholanthrene in C57BL/6 mice (Shu et al., 1985). The assays described here are applicable for other cell lines
  4. DMEM high glucose (Thermo Fisher Scientific, GibcoTM, catalog number: 11965092 , or GE Healthcare, HyCloneTM , catalog number: SH30022 )
  5. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 16000044 )
  6. 100 mM sodium pyruvate solution (Thermo Fisher Scientific, GibcoTM, catalog number: 11360070 )
  7. HEPES, 1 M buffer solution (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
  8. Penicillin and streptomycin
  9. Mitoxantrone (Sigma-Aldrich, catalog number: M6545 )
  10. Trypsin (0.25%) (Thermo Fisher Scientific, GibcoTM, catalog number: 15050065 )
  11. 1x PBS, pH 7.4 (Thermo Fisher Scientific, GibcoTM, catalog number: 10010031 )
  12. DiOC6(3) (3,3’-dihexyloxacarbocyanine Iodide) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D273 )
  13. DAPI (4’,6-diamidino-2-phenylindole, dihydrochloride) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D1306 )
  14. CALR antibody (Ab): rabbit monoclonal Ab, clone number: EPR3924 (Abgent, catalog number: AJ1124a ; or Abcam, catalog number: ab92516 ); or rabbit polyclonal Ab (Abcam, catalog number: ab2907 )
  15. Alexa488 conjugated goat anti-rabbit IgG (H+L) secondary antibody (Thermo Fisher Scientific, Invitrogen, catalog number: A-11008 )
  16. ATP Bioluminescent Assay Kit (Sigma-Aldrich, catalog number: FL-AA ) or ENLITEN® ATP Assay System (Promega, catalog number: FF2000 )
  17. Annexin V Apoptosis Detection Kit (BD, catalog number: 559763 )
  18. HMGB1 ELISA Kit (Tecan Trading, catalog number: ST51011 )
  19. 1x Annexin V binding buffer (see Recipes)
  20. ATP assay mix (see Recipes)
  21. rLuciferase/Luciferin buffer(see Recipes)

Equipment

  1. Centrifuge
  2. Water-Jacketed CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: HERAcell® 150i )
  3. Multi-channel pipette
  4. SpectraMax L microplate reader (Molecular Device, model: SpectraMax L)
  5. SpectraMax i3 multi-mode detection platform (Molecular Device, model: SpectraMax i3)
  6. AttuneTM NxT flow cytometer (Thermo Fisher Scientific, model: AttuneTM NxT Flow Cytometer )

Note: Alternatively, VICTORTM X multilabel reader (PerkinElmer) and MACSQuant® Analyzer 10 (Miltenyi Biotec) are also suitable.

Software

  1. Excel (Microsoft Office)
  2. GraphPad Prism (San Diego, CA, USA)
  3. FlowJo software (Treestar Inc., Ashland, OR, USA)

Procedure

MCA205 tumor cells are cultured in DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, 10 mM HEPES and 100 U/ml penicillin and streptomycin. Cells are seeded in 24 well plates (5 x 104 cells in 500 µl culture medium per well). The next day, the culture medium is replaced carefully with 500 µl pre-warmed fresh medium (with or without chemotherapeutic drugs). Culture media can be substituted according to the requirements of different cell lines. The recommended dose for mitoxantrone is 1-10 µM.

  1. Detection of cell death
    1. At desired time points, the supernatant is collected thoroughly by pipette. Adherent cells are detached and dispersed with trypsin. All cells are then collected to combine with the original supernatant.
    2. Cells are collected in 2 ml Eppendorf tubes by spinning at 300 x g, 10 min, 4 °C and the supernatant is discarded. Do not disturb the cell pellets while removing the supernatant.
    3. Cell pellets are resuspended in PBS containing 1 µM DiOC6(3), incubated at 37 °C for 15 min. The stained cells are washed twice with PBS (10 times the initial volume) and then resuspended in 400 µl PBS containing 1 µg/ml DAPI. The samples are ready for cytofluorometry analysis within 30 min (Figure 1).
    4. Alternatively, cell pellets are washed with PBS twice and resuspended in 100 µl 1x Annexin V binding buffer containing PE-Annexin V (1:50 dilution) and DAPI (1 µg/ml). The cell concentration should not exceed 1 x 106 cells/ml. After incubation for 15 min at room temperature in the dark, 400 µl 1x Annexin V binding buffer are added in each tube. The samples are ready for cytofluorometry analysis within 30 min (Figure 2).


      Figure 1. Typical dot plots of DiOC6(3)-DAPI double staining with MCA205 tumor cells that have been pre-treated with PBS control (left) or 1 µM mitoxantrone, 28 h (right)


      Figure 2. Typical dot plots of Annexin V PE-DAPI double staining with MCA205 tumor cells that have been pre-treated with PBS control (left) or 1 µM mitoxantrone, 24 h (right)

  2. Detection of calreticulin (CALR) exposure
    1. For each sample, the entire well of supernatant and cells are collected and combined in the same Eppendorf tube at desired time points (e.g., 6 h, 16 h, 24 h and 48 h). Discard the supernatant after spinning at 300 x g, 10 min at 4 °C.
    2. Tumor cells are stained with anti-CALR rabbit monoclonal Ab (1:500 dilution, Abgent, clone EPR3924) or rabbit polyclonal CALR Ab (used at dilution 1:100, Abcam) on ice for 30 min in the dark.
    3. After a wash with PBS (10 times the initial volume), tumor cells are stained with Alexa488 conjugated goat anti-rat IgG (H+L) secondary antibody (1:500 dilution) on ice for 30 min.
    4. After wash with PBS (10 times the initial volume), tumor cells are further stained with DAPI at 1 µg/ml, 5 min at room temperature. All samples are kept on ice and analyzed by cytofluorometry within 30 min. To calculate CALR+cells among live cells, DAPI+cells are excluded from the gating (Figure 3).


      Figure 3. Typical dot plots of CALR-DAPI double staining with MCA205 tumor cells that have been pre-treated with PBS control (left) or 1 µM mitoxantrone, 24 h (right)

  3. Detection of ATP release
    1. Three hundred microliters (300 µl) of the supernatant is collected at desired time points and spun at 500 x g, 5 min at 4 °C to remove the inclusion of cells or debris.
    2. The collected supernatant should be subjected to ATP bioluminescent assay immediately or stored at -80 °C (150 µl per aliquot). Storage of supernatant at room temperature or repeated freeze/thaw lead to rapid ATP degradation. There’s no need to spin supernatant samples again after thawing.
    3. For ATP Bioluminescent Assay Kit (Sigma-Aldrich), a 25-fold dilution of ATP Assay Mix stock solution with ATP Assay Mix Dilution Buffer is recommended. The samples or standards (100 µl per well) are added into the plate containing diluted assay mix (100 µl per well) with multi-channel pipette rapidly.
    4. For ENLITEN® ATP Assay System (Promega), the samples and standards (20 µl/well) are added into the plate on ice. Reconstituted rLuciferase/Luciferin Reagent (100 µl/well) are then added into each well with multi-channel pipette rapidly.
    5. ATP-driven chemiluminescence signals are recorded with a luminescence microplate reader. ATP concentrations in samples are calculated with standard curves (ranging from 10-6 to 10-12 with a 10 fold serial dilution) (Figure 4).
      Caution: Microplates should be scanned immediately after samples are mixed with assay mix.


      Figure 4. A typical standard curve for calculating ATP content

  4. Detection of HMGB1 release
    1. Three hundred microliters (300 µl) of the supernatant is collected at desired time points and spun at 500 x g, 5 min at 4 °C to remove the inclusion of cells or debris.
    2. The supernatant is either applied to ELISA-based quantification freshly, or stored at -80 °C (150 µl per aliquot). Repeated freeze/thaw of samples should be avoided. The concentration of HMGB1 is detected following user’s instruction with standard test procedures.
    3. Incubation of samples and standards with pre-coated plate at 37 °C for 20-24 h is necessary to reach the maximal sensitivity. High sensitive detection procedures can be applied to detect low HMGB1 content in the samples.
    4. Optical density is measured with a photometer at 450 nm and 600 nm (used as the reference, subtracted from the 450 nm reading). The concentration of HMGB1 is calculated according to the standard curve (Figure 5).


      Figure 5. A typical standard curve for calculating HMGB1 content

Data analysis

Flow cytometry data can be analyzed by flowjo software (Treestar Inc., Ashland, OR, USA). Apoptotic cells are DAPI-DiOC6(3)low or DAPI-Annexin V+. Necrotic cells are DAPI+DiOC6(3)low or DAPI+Annexin V+. Data should be calculated and presented as mean ± SD (or SEM). Cells with CALR exposure are CALR+DAPI-. The concentration of ATP and HMGB1 are calculated according to standard curves. It is recommended that all experiments should be performed at least 2-3 times independently. Statistics and data visualization are generated by Graphpad Prism software (San Diego, CA, USA). Unpaired, two-tailed Student’s t-test is the preferred method.

Notes

  1. To compare the cytotoxicity of drugs or the susceptibility of different cells, cell death can be checked at various time points, or in response to different doses of drugs.
  2. To accurately detect ATP in the supernatant, all procedures should be performed on ice as quickly as possible.
  3. It is possible to detect ATP from frozen supernatant, which should have been stored at -80 °C preferentially. However, special attentions should be paid during supernatant collection. Cell contamination in the supernatant will give extremely high level of ATP, due to cell rupture after freeze/thaw. The tips of pipette should not reach the bottom of culture plate. Prevent disturbances of the media and collect the supernatant gently.
  4. Annexin V-PE is more sensitive and reliable than Annexin V-FITC kits to detect cell death.

Recipes

  1. 1x Annexin V binding buffer
    Dilute 10x Annexin V binding buffer provided in Annexin V Apoptosis Detection Kit with ddH2O
  2. ATP assay mix
    For ATP Bioluminescent Assay Kit, ATP assay mix and ATP assay mix dilution buffer are dissolved with 5 ml and 50 ml ddH2O respectively on ice for 30 min. Dilute the ATP assay mix stock solution with 25-fold dilution buffer.
  3. rLuciferase/Luciferin buffer
    For ENLITEN® ATP Assay System, rLuciferase/Luciferin is reconstituted with 12 ml reconstitution buffer provided in the kit.

Acknowledgments

Dr. Yuting Ma is supported by Chinese National Thousand Young Talents Program, Chinese Academy of Medical Sciences research grant (Grant No. 2015RC310003) and Natural Science Foundation of Jiangsu Province, China (Grant No. BK20160379).

References

  1. Ma, Y., Adjemian, S., Mattarollo, S. R., Yamazaki, T., Aymeric, L., Yang, H., Portela Catani, J. P., Hannani, D., Duret, H., Steegh, K., Martins, I., Schlemmer, F., Michaud, M., Kepp, O., Sukkurwala, A. Q., Menger, L., Vacchelli, E., Droin, N., Galluzzi, L., Krzysiek, R., Gordon, S., Taylor, P. R., Van Endert, P., Solary, E., Smyth, M. J., Zitvogel, L. and Kroemer, G. (2013). Anticancer chemotherapy-induced intratumoral recruitment and differentiation of antigen-presenting cells. Immunity 38(4): 729-741.
  2. Ma, Y., Mattarollo, S. R., Adjemian, S., Yang, H., Aymeric, L., Hannani, D., Portela Catani, J. P., Duret, H., Teng, M. W., Kepp, O., Wang, Y., Sistigu, A., Schultze, J. L., Stoll, G., Galluzzi, L., Zitvogel, L., Smyth, M. J. and Kroemer, G. (2014). CCL2/CCR2-dependent recruitment of functional antigen-presenting cells into tumors upon chemotherapy. Cancer Res 74(2): 436-445.
  3. Shu, S. Y. and Rosenberg, S. A. (1985). Adoptive immunotherapy of newly induced murine sarcomas. Cancer Res 45(4): 1657-1662.
  4. Yang, H., Yamazaki, T., Pietrocola, F., Zhou, H., Zitvogel, L., Ma, Y. and Kroemer, G. (2015). STAT3 inhibition enhances the therapeutic efficacy of immunogenic chemotherapy by stimulating type 1 interferon production by cancer cells. Cancer Res 75(18): 3812-3822.

简介

积累的证据表明免疫系统在癌症治疗中的重要作用。某些化学治疗药物可以有效诱导伴随癌细胞死亡的“细胞死亡相关分子模式”(CDAMPs)的释放。 CDAMP可以与免疫细胞上的相应受体结合并刺激免疫应答以实现长期的肿瘤控制(Ma等,2013; Ma等人,2014; Yang& em>等,,2015)。在所报道的CDAMP中,钙网蛋白(CALR),ATP和HMGB1是众所周知的免疫刺激作用。在这里我们描述我们应用于测量细胞死亡和这些CDAMPs的测定。简单地说,细胞死亡可以通过4',6-二脒基-2-苯基吲哚(DAPI)与3,3'-二氧基氧羰基花青氨基碘[DiOC6(3)]或Annexin V的共染色来分析。通过流式细胞术检测。 ATP和HMGB1释放可以通过发光测定和ELISA测定分别进行定量。

背景 乳酸脱氢酶测定和台盼蓝染色是检测细胞死亡的传统方法。我们在这里描述两种可行和经济的解决方案,以通过流式细胞术(FCM)检测凋亡和坏死细胞死亡。 DAPI标记具有破坏完整性(坏死)的细胞,而膜联蛋白V结合磷脂酰丝氨酸(其在凋亡后被外化)。 DiOC6(3)摄取表明线粒体跨膜电位(MTP),MTP的崩解显示凋亡。 DAPI不需要补偿藻红蛋白(PE,其与膜联蛋白V蛋白结合)或DiOC6(3),因此在这些测定中显示优于碘化丙啶(PI)的优点。
  CALR是内质网蛋白,胱天蛋白酶级联的激活引发CALR移位到细胞质膜。可以通过用相应抗体的免疫荧光染色检测CALR暴露,然后进行基于FCM或显微镜检测。细胞外ATP可以通过荧光素酶的酶活性测定,而HMGB1浓度可以通过敏感ELISA试剂盒检测。

关键字:细胞死亡, DiOC6(3), 膜联蛋白V, 钙网蛋白, HMGB1, ATP, 荧光素酶

材料和试剂

  1. 24孔板
  2. 2 ml Eppendorf管
  3. 在C57BL/6小鼠中由3-甲基胆蒽诱导MCA205纤维肉瘤细胞(H2 b )(Shu等人,1985)。这里描述的测定适用于其他细胞系
  4. DMEM高葡萄糖(Thermo Fisher Scientific,Gibco TM,目录号:11965092或GE Healthcare,HyClone TM,目录号:SH30022)
  5. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:16000044)
  6. 100mM丙酮酸钠溶液(Thermo Fisher Scientific,Gibco TM,目录号:11360070)
  7. HEPES,1M缓冲溶液(Thermo Fisher Scientific,Gibco TM,目录号:15630080)
  8. 青霉素和链霉素
  9. 米托蒽醌(Sigma-Aldrich,目录号:M6545)
  10. 胰蛋白酶(0.25%)(Thermo Fisher Scientific,Gibco TM,目录号:15050065)
  11. 1×PBS,pH 7.4(Thermo Fisher Scientific,Gibco TM,目录号:10010031)
  12. DiOC6(3)(3,3'-二己氧基亚硫氰酸碘化物)(Thermo Fisher Scientific,Molecular Probes TM,目录号:D273)
  13. DAPI(4',6-二脒基-2-苯基吲哚,二盐酸盐)(Thermo Fisher Scientific,Molecular Probes TM,目录号:D1306)
  14. CALR抗体(Ab):兔单克隆Ab,克隆号:EPR3924(Abgent,目录号:AJ1124a;或Abcam,目录号:ab92516);或兔多克隆Ab(Abcam,目录号:ab2907)
  15. Alexa488缀合的山羊抗兔IgG(H + L)二抗(Thermo Fisher Scientific,Invitrogen,目录号:A-11008)
  16. ATP生物发光测定试剂盒(Sigma-Aldrich,目录号:FL-AA)或ENLITEN ATP测定系统(Promega,目录号:FF2000)
  17. Annexin V细胞凋亡检测试剂盒(BD,目录号:559763)
  18. HMGB1 ELISA试剂盒(Tecan Trading,目录号:ST51011)
  19. 1x Annexin V结合缓冲液(见配方)
  20. ATP测定组合(参见食谱)
  21. rLuciferase/Luciferin缓冲液(参见食谱)

设备

  1. 离心机
  2. 水包覆的CO 2培养箱(Thermo Fisher Scientific,Thermo Scientific,superson TM,型号:HERAcell 150i)
  3. 多通道移液器
  4. SpectraMax L酶标仪(Molecular Device,型号:SpectraMax L)
  5. SpectraMax i3多模式检测平台(分子装置,型号:SpectraMax i3)
  6. Attune TM NxT流式细胞仪(Thermo Fisher Scientific,型号:Attune TM NxT Flow Cytometer)

注意:或者,VICTOR X multilabel阅读器(PerkinElmer)和MACSQuant /em>分析仪10(Miltenyi Biotec)也是合适的。

软件

  1. Excel(Microsoft Office)
  2. GraphPad Prism(美国加利福尼亚州圣地亚哥)
  3. FlowJo软件(Treestar Inc.,Ashland,OR,USA)

程序

将MCA205肿瘤细胞在补充有10%FBS,1mM丙酮酸钠,10mM HEPES和100U/ml青霉素和链霉素的DMEM中培养。将细胞接种在每孔500μl培养基的24孔板(5×10 4个细胞)中。第二天,用500μl预热的新鲜培养基(含或不含化疗药物)仔细替换培养基。培养基可根据不同细胞系的要求进行取代。米托蒽醌的推荐剂量为1-10μM。

  1. 检测细胞死亡
    1. 在所需时间点,通过移液管彻底收集上清液。贴壁细胞被分离并用胰蛋白酶分散。然后收集所有细胞以与原始上清液组合。
    2. 通过在300×g,10分钟,4℃下旋转将细胞收集在2ml Eppendorf管中,并弃去上清液。去除上清液时请勿打扰细胞沉淀
    3. 将细胞沉淀重新悬浮于含有1μMDiOC6(3)的PBS中,37℃孵育15分钟。将染色的细胞用PBS洗涤两次(10倍初始体积),然后重悬于含有1μg/ml DAPI的400μlPBS中。样品准备好在30分钟内进行细胞荧光分析(图1)
    4. 或者,用PBS洗涤细胞沉淀两次并重悬于含有PE-膜联蛋白V(1:50稀释)和DAPI(1μg/ml)的100μl1x Annexin V结合缓冲液中。细胞浓度不应超过1×10 6细胞/ml。在黑暗中室温孵育15分钟后,在每个管中加入400μl1x Annexin V结合缓冲液。样品准备在30分钟内进行细胞荧光测定分析(图2)

      图1.用PBS对照(左)或1μM米托蒽醌预处理的MCA205肿瘤细胞的DiOC6(3)-DAPI双染色的典型点图,28小时(右)


      图2.具有用PBS对照(左)或1μM米托蒽醌预处理的MCA205肿瘤细胞的膜联蛋白V PE-DAPI双染色的典型点图,24小时(右)
  2. 钙网蛋白(CALR)暴露的检测
    1. 对于每个样品,收集上清液和细胞的整个孔并在所需时间点(例如6小时,16小时,24小时和48小时)在相同的Eppendorf管中合并。以300×g旋转后弃去上清液,4℃10分钟
    2. 使用抗CALR兔单克隆抗体(1:500稀释,Abgent,克隆EPR3924)或兔多克隆CALR Ab(稀释1:100,Abcam)在黑暗中在冰上对30分钟染色肿瘤细胞。
    3. 用PBS洗涤(初始体积的10倍)后,在冰上用Alexa488缀合的山羊抗大鼠IgG(H + L)二抗(1:500稀释)将肿瘤细胞染色30分钟。
    4. 用PBS洗涤(10倍初始体积)后,肿瘤细胞用DAPI以1μg/ml进一步染色,室温5分钟。将所有样品保存在冰上,并在30分钟内通过细胞荧光测定法进行分析。为了在活细胞中计算CALR + 细胞,从门控中排除DAPI + 细胞(图3)。


      图3.使用PBS对照(左)或1μM米托蒽醌预处理的MCA205肿瘤细胞的CALR-DAPI双染色的典型点图,24小时(右)

  3. 检测ATP释放
    1. 在期望的时间点收集300微升(300μl)上清液,并在5℃下以5℃旋转5分钟以除去细胞或碎屑。
    2. 收集的上清液应立即进行ATP生物发光测定或储存于-80℃(每等分试样150μl)。上清液在室温或反复冻融过程中的存储导致ATP降解迅速。解冻后,无需再次上清样品。
    3. 对于ATP生物发光测定试剂盒(Sigma-Aldrich),推荐使用ATP Assay Mix稀释缓冲液进行25倍稀释的ATP Assay Mix储备溶液。将样品或标准品(每孔100μl)快​​速加入含有稀释的测定混合物(100μl/孔)的板中。
    4. 对于ENLITEN ® ATP测定系统(Promega),将样品和标准品(20μl/孔)加入到冰上的板中。然后将重组的rLuciferase /萤光素试剂(100μl/孔)快速加入每个孔中,多孔移液管。
    5. 用发光酶标仪记录ATP驱动的化学发光信号。使用10倍连续稀释度的标准曲线(范围从10℃至10℃至10℃-12℃)计算样品中的ATP浓度(图4)。
      注意:样品与试剂盒混合后,应立即扫描微孔板。


      图4.计算ATP内容的典型标准曲线

  4. 检测HMGB1释放
    1. 在期望的时间点收集300微升(300μl)上清液,并在5℃下以5℃旋转5分钟以除去细胞或碎屑。
    2. 将上清液新鲜施用于基于ELISA的定量,或储存在-80℃(每等分试样150μl)。应避免重复冻结/解冻样品。 HMGB1的浓度是按照用户使用的标准测试程序进行的。
    3. 样品和标准品在37℃下孵育20-24小时是必要的,以达到最大的灵敏度。高灵敏度检测程序可用于检测样品中的低HMGB1含量
    4. 光密度用450nm和600nm的光度计测量(用作参考,从450nm读数中减去)。 HMGB1的浓度根据标准曲线计算(图5)

      图5.计算HMGB1内容的典型标准曲线

数据分析

流式细胞术数据可以通过flowjo软件(Treestar Inc.,Ashland,OR,USA)进行分析。细胞凋亡细胞是DAPI(DiC)(3) low 或DAPI Annexin V + 。坏死细胞是DAPI + DiOC6(3) low 或DAPI + Annexin V + 。数据应以平均值±SD(或SEM)计算并显示。具有CALR暴露的细胞是CALR + DAPI - 。 ATP和HMGB1的浓度根据标准曲线计算。建议所有实验应至少2-3次独立进行。统计和数据可视化由Graphpad Prism软件(San Diego,CA,USA)生成。不配对的双尾学生的测试是首选方法。

笔记

  1. 为了比较药物的细胞毒性或不同细胞的易感性,可以在不同的时间点或针对不同剂量的药物检查细胞死亡。
  2. 为了准确检测上清液中的ATP,所有操作都应尽快在冰上进行
  3. 可以从冷冻上清液中检测出ATP,这些蛋白质应优先储存在-80°C。但是,在收集上清液时应特别注意。上清液中的细胞污染会由于细胞在冻融后破裂而产生极高的ATP含量。移液管的尖端不能到达培养板的底部。防止介质干扰,并轻轻收集上清液。
  4. 膜联蛋白V-PE比Annexin V-FITC试剂盒更灵敏和可靠,以检测细胞死亡。

食谱

  1. 1x Annexin V结合缓冲液
    稀释10倍膜联蛋白V结合缓冲液提供的Annexin V细胞凋亡检测试剂盒与ddH 2 O
  2. ATP测定混合物
    对于ATP生物发光测定试剂盒,ATP测定混合物和ATP测定混合物稀释缓冲液分别用5ml和50ml ddH 2 O在冰上溶解30分钟。用25倍稀释缓冲液稀释ATP测定混合物储备溶液。
  3. 荧光素酶/萤光素缓冲液
    对于ENLITEN ® ATP测定系统,使用试剂盒中提供的12ml重组缓冲液重构rLuciferase/Luciferin。

致谢

马玉泰博士获得中国医学科学院中国千年青年人才计划资助(授权号:2015RC310003)和江苏省自然科学基金资助(批准号:BK20160379)。

参考文献

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  2. Ma,Y.,Mattarollo,SR,Adjemian,S.,Yang,H.,Aymeric,L.,Hannani,D.,Portela Catani,JP,Duret,H.,Teng,MW,Kepp,O.,Wang, Y.,Sistigu,A.,Schultze,JL,Stoll,G.,Galluzzi,L.,Zitvogel,L.,Smyth,MJ和Kroemer,G。(2014)。< a class ="ke-insertfile"化学治疗后功能性抗原呈递细胞向肿瘤中CCL2/CCR2依赖性募集的指标是"href ="http://www.ncbi.nlm.nih.gov/pubmed/24302580"target ="_ blank" > Cancer Res 74(2):436-445。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ma, Y. and Yang, H. (2016). In vitro Assays for the Detection of Calreticulin Exposure, ATP and HMGB1 Release upon Cell Death. Bio-protocol 6(24): e2076. DOI: 10.21769/BioProtoc.2076.
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