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The Stat (Signal Transducer and Activator of Transcription) family of proteins are critical signal transducers involved in fundamental cellular processes, including cell growth and differentiation, development, apoptosis, immune responses and inflammation. In here, we describe a simple and reproducible flow cytometry protocol to measure Stat protein phosphorylation in splenocyte preparations from malaria infected mice.

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Intracellular Staining for Phosphorylated STAT4 and STAT5 in Mouse Splenocytes
小鼠脾脏细胞中磷酸化STAT4和STAT5的胞内染色

免疫学 > 免疫细胞染色 > 流式细胞术
作者: Ana Villegas-Mendez
Ana Villegas-MendezAffiliation: Department of Immunology and Infection, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK
Bio-protocol author page: a1147
J. Brian de Souza
J. Brian de SouzaAffiliation: Department of Immunology and Infection, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK
Bio-protocol author page: a1151
Seen-Wai Lavelle
Seen-Wai LavelleAffiliation: Department of Immunology and Infection, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK
Bio-protocol author page: a1152
Emily Gwyer Findlay
Emily Gwyer FindlayAffiliation: Department of Immunology and Infection, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK
Bio-protocol author page: a1153
Tovah N. Shaw
Tovah N. ShawAffiliation: Department of Immunology and Infection, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK
Bio-protocol author page: a1154
Christiaan J. Saris
Christiaan J. SarisAffiliation: Department of Inflammation Research, Amgen, Inc., Thousand Oaks, CA, USA
Bio-protocol author page: a1155
Christopher A. Hunter
Christopher A. HunterAffiliation: Department of Pathobiology, University of Pennsylvania, Philadelphia, USA
Bio-protocol author page: a1156
Eleanor M. Riley
Eleanor M. Riley Affiliation: Department of Immunology and Infection, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK
Bio-protocol author page: a1157
 and Kevin N. Couper
Kevin N. CouperAffiliation: Department of Immunology and Infection, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK
For correspondence: kevin.couper@manchester.ac.uk
Bio-protocol author page: a1158
Vol 4, Iss 3, 2/5/2014, 3795 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1043

[Abstract] The Stat (Signal Transducer and Activator of Transcription) family of proteins are critical signal transducers involved in fundamental cellular processes, including cell growth and differentiation, development, apoptosis, immune responses and inflammation. In here, we describe a simple and reproducible flow cytometry protocol to measure Stat protein phosphorylation in splenocyte preparations from malaria infected mice.

[Abstract]

Materials and Reagents

  1. C57BL/6 mice
  2. RBC lysing buffer (BD Biosciences, catalog number: 555899 )
  3. FACS buffer: Hanks balance salt solution (HBSS) with 2% foetal calf serum (FCS)
  4. Trypan blue (Sigma-Aldrich, catalog number: T8154 )
  5. AIM V® Medium (Life Technologies, catalog number: 31035025 )
  6. Recombinant IL-2 (eBioscience) (stock solutions prepared as recommended by manufacturer)br />
  7. Recombinant IL-12 (R&D Systems) (stock solutions prepared as recommended by manufacturer)
  8. 4% paraformaldehyde
  9. 90% ice-cold methanol
  10. CD4 antibody (GK1.5) (eBioscience)
  11. CD44 antibody (IM7) (eBioscience)
  12. CD62L antibody (MEL-14) (eBioscience)
  13. T-bet (4B10) antibody (eBioscience)
  14. Phosphorylated STAT4 antibody (at residue Y693, clone 38) (BD Biosciences)
  15. Phosphorylated STAT5 antibody (at residue Y694, clone 47) (BD Biosciences)

Equipment

  1. 6-well plates
  2. 70 μm cell strainer (BD Biosciences)
  3. Haemocytometer
  4. Refrigerated table top centrifuge
  5. LSR II (BD Systems)

Procedure

  1. Spleens were obtained from naive and malaria-infected C57BL/6 mice and were homogenized at room temperature in 6-well plates with 6 ml of HBSS with 2% FCS through a 70 μm cell strainer to form single-cell suspensions.
  2. Red blood cells were lysed using 1 to 2 ml of RBC lysing buffer (depending on size of spleen) and splenocytes were washed once at 200 x g 4 °C for 5 min with cold FACS buffer (HBSS with 2% FCS).
  3. Splenocytes were resuspended by gentle tapping on a rack in FACS buffer and kept on ice at all times to avoid background phosphorylation of STAT proteins.
  4. Viability and cell counts were obtained by trypan blue exclusion using a haemocytometer.
  5. Cells were washed once with 1 ml AIM V® Medium, resuspended at 20 x 106 cells/ml and rested on ice for a minimum of 20 min.
  6. 1 × 106 cells were incubated with 20 ng/ml rIL-2 or 2.5 ng/ml rIL-12 for 10 min at 37 °C, 5% CO2 (final volume of 200 μl) and immediately fixed on ice for 15 min by adding an equal volume of 4% paraformaldehyde.
  7. Cells were washed with FACS buffer, resuspended in 500 μl of 90% ice-cold methanol and immediately stored down at -20 °C for a minimum of 2 h (cells can be kept for up to a month without affecting further staining).
  8. Splenocytes were washed twice with FACS buffer and stained in FACS buffer at room temperature for 30 min for CD4 (GK1.5), CD44 (IM7), CD62L (MEL-14), T-bet (4B10) and phosphorylated STAT4 (at residue Y693, clone 38) or phosphorylated STAT5 (at residue Y694, clone 47).
  9. Cells were washed with FACS buffer and analysed by flow cytometry.
  10. Fluorescence minus one controls were included to validate flow cytometric results. Flow cytometry acquisition was performed using an LSR II.

Acknowledgments

This protocol is adapted from Villegas-Mendez et al. (2013).

References

  1. Villegas-Mendez, A., de Souza, J. B., Lavelle, S. W., Gwyer Findlay, E., Shaw, T. N., van Rooijen, N., Saris, C. J., Hunter, C. A., Riley, E. M. and Couper, K. N. (2013). IL-27 receptor signalling restricts the formation of pathogenic, terminally differentiated Th1 cells during malaria infection by repressing IL-12 dependent signals. PLoS Pathog 9(4): e1003293.

材料和试剂

  1. C57BL/6小鼠
  2. RBC裂解缓冲液(BD Biosciences,目录号:555899)
  3. FACS缓冲液:含有2%胎牛血清(FCS)的Hanks平衡盐溶液(HBSS)
  4. 台盼蓝(Sigma-Aldrich,目录号:T8154)
  5. AIM V ® Medium(Life Technologies,目录号:31035025)
  6. 重组IL-2(eBioscience)(如制造商推荐制备储备溶液)。
  7. 重组IL-12(R& D Systems)(如制造商推荐制备储备溶液)
  8. 4%多聚甲醛
  9. 90%冰冷的甲醇
  10. CD4抗体(GK1.5)(eBioscience)
  11. CD44抗体(IM7)(eBioscience)
  12. CD62L抗体(MEL-14)(eBioscience)
  13. T-bet(4B10)抗体(eBioscience)
  14. 磷酸化的STAT4抗体(在残基Y693,克隆38)(BD Biosciences)
  15. 磷酸化的STAT5抗体(在残基Y694,克隆47)(BD Biosciences)

设备

  1. 6孔板
  2. 70μm细胞过滤器(BD Biosciences)
  3. 血细胞计数器
  4. 冷冻台式离心机
  5. LSR II(BD系统)

程序

  1. 从幼稚和疟疾感染的C57BL/6小鼠获得脾脏,并在6孔板中用6ml具有2%FCS的HBSS通过70μm细胞过滤器在室温下匀浆以形成单细胞悬浮液。 />
  2. 使用1至2ml的RBC裂解缓冲液(取决于脾的大小)裂解红细胞,并且将脾细胞在200×g 4℃下用冷FACS缓冲液(HBSS用2 %FCS)。
  3. 通过在FACS缓冲液中轻轻敲击架子来重悬脾细胞,并一直保持在冰上以避免STAT蛋白的背景磷酸化。
  4. 使用血细胞计数器通过台盼蓝排除获得存活力和细胞计数。
  5. 将细胞用1ml AIM V medium洗涤一次,以20×10 6个细胞/ml重悬,并在冰上搁置最少20分钟。
  6. 将1×10 6个细胞与20ng/ml rIL-2或2.5ng/ml rIL-12在37℃,5%CO 2下孵育10分钟( 最终体积为200μl),立即通过加入等体积的4%多聚甲醛在冰上固定15分钟。
  7. 用FACS缓冲液洗涤细胞,重悬于500μl90%冰冷的甲醇中,并立即在-20℃下储存至少2小时(细胞可以保存长达一个月,而不影响进一步的染色)。
  8. 脾细胞用FACS缓冲液洗涤两次并在室温下在FACS缓冲液中染色CD4(GK1.5),CD44(IM7),CD62L(MEL-14),T-bet(4B10)和磷酸化STAT4 Y693,克隆38)或磷酸化的STAT5(在残基Y694,克隆47)。
  9. 用FACS缓冲液洗涤细胞并通过流式细胞术分析。
  10. 包括荧光减去一个对照以验证流式细胞术结果。 使用LSR II进行流式细胞术采集。

致谢

该协议改编自Villegas-Mendez等人(2013)。

参考文献

  1. Villegas-Mendez,A.,de Souza,JB,Lavelle,SW,Gwyer Findlay,E.,Shaw,TN,van Rooijen,N.,Saris,CJ,Hunter,CA,Riley,EMand Couper, 。 IL-27受体信号传导通过抑制IL在疟疾感染期间限制致病性终末分化的Th1细胞的形成 -12依赖信号。 PLoS Pathog 9(4):e1003293。
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Villegas-Mendez, A., de Souza, J. B., Lavelle, S., Findlay, E. G., Shaw, T. N., Saris, C. J., Hunter, C. A., Riley, E. M. and Couper, K. N. (2014). Intracellular Staining for Phosphorylated STAT4 and STAT5 in Mouse Splenocytes. Bio-protocol 4(3): e1043. DOI: 10.21769/BioProtoc.1043; Full Text
  2. Villegas-Mendez, A., de Souza, J. B., Lavelle, S. W., Gwyer Findlay, E., Shaw, T. N., van Rooijen, N., Saris, C. J., Hunter, C. A., Riley, E. M. and Couper, K. N. (2013). IL-27 receptor signalling restricts the formation of pathogenic, terminally differentiated Th1 cells during malaria infection by repressing IL-12 dependent signals. PLoS Pathog 9(4): e1003293.




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