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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, 2691 views, 0 Q&A, How to cite
DOI: http://dx.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.

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.


How to cite this protocol: 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



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