Application Information Bulletin: Cell Signaling

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1 Application Information Bulletin: Cell Signaling Detection of Both Surface Proteins and Intracellular Phospho-Determinants in Whole Blood Using the PerFix-p Reagent System William L. Godfrey 1, Li Yang 1, Samantha Bauchiero 1, James W. Tung 1, Valerie Mallet 2, Lidice Lopez 1, Vincent Shankey 1, Emmanuel Gautherot 2 Beckman Coulter Inc, 1 Miami, FL, USA; 2 Marseille, FRANCE

2 Detection of Both Surface Proteins and Intracellular Phospho-Determinants in Whole Blood Using the PerFix-p Reagent System ABSTRACT Introduction. Flow cytometry provides a unique methodology to monitor signaling pathways, allowing a coordinated measurement of multiple phospho-epitopes in the context of cell surface markers. Application of this technology has been hindered by the diversity of sample preparation protocols designed to unmask particular epitopes, often at the expense of surface marker detection. A novel sample preparation methodology, the PerFix-p* reagent system (Beckman Coulter), allows simultaneous and reproducible detection of multiple phospho-epitopes in conjunction with surface markers on a variety of samples, including whole blood, peripheral blood mononuclear cells, and bone marrow. The ability to multiplex phospho-epitope detection with immunophenotyping opens new possibilities to map changes in the phosphorylation pathways associated with cellular processes in normal and abnormal cells. Methods. Whole blood was activated with either lipopolysaccharide (LPS) or granulocyte/monocyte colony stimulating factor (GM-CSF) before processed with PerFix-p fixative and lysis buffers. Untreated whole blood was also processed as a control. Samples were then stained for surface and intracellular markers, followed by resuspension in a stabilizing buffer. Data from these samples were collected on a Gallios* cytometer and analyzed with Kaluza* software (Beckman Coulter). Results. Following LPS activation, the phosphorylation changes in p38 [T180/Y182], ERK [T202/Y204], AKT [S473], and S6 [S235/236] in monocytes (CD45 + CD14 + ) can be detected using PerFix-p reagents. For example, we observed a thirteen-fold increase of phospho-erk signal in LPS-stimulated vs. untreated whole blood. We also observed a twenty-seven fold up-regulation in phospho-s6 signal and a more than 60-fold augmentation of phospho-p38 level. The detection of the changes in these markers using the PerFix-p reagents were done free of methanol, which shortens the processing time. Nuclear phospho-stat5 [Y694] can be detected in GM-CSF-activated blood samples, but requires an additional treatment. A number of cell surface epitopes, such as CD14, are sensitive to methanol fixation. However, CD14 can be readily detected with the PerFix-p reagents by staining this surface marker prior to methanol treatment. The phospho-stat5 staining, on the other hand, is performed after the treatment. This approach enables the detection of difficult intranuclear signaling determinants (for instance, phospho-stat5) without sacrificing staining of the surface molecules. Conclusion. The PerFix-p sample preparation approach allows reliable and reproducible detection of multiplexed immunophenotyping and intracellular phospho-epitopes in a range of samples, including whole blood and bone marrow. As a result, the PerFix-p reagent kit brings a powerful new tool for studying the phospho-signaling network. 1

3 Phospho-protein signalling pathways employed GM-CSF Toll-like-receptor Jakstat_pathway.svg LPS (lipopolysaccharide) is known to activate multiple signaling pathways in peripheral blood monocytes via the Toll-Like Receptor 4 complex. Following LPS exposure, monocytes rapidly activate all three major MAP Kinase pathways (ERK, p38, and SAP/JNK) in addition to PI3 Kinase and IKK/NFkB pathways. JAK/STAT pathways mediate signaling from a number of membrane receptors and are activated by GM-CSF, leading to phosphorylation of STAT5. 2

4 MATERIALS & METHODS Whole blood was activated with either lipopolysaccharide (LPS) or granulocyte/monocyte colony stimulating factor (GM-CSF) before processed with PerFix-p fixative and lysis buffers as shown in the flow charts below. Samples were also prepared with FIX&PERM (Invitrogen, Carlsbad, CA) and Phosflow Lyse/Fix / Perm Buffer III / Stain (BD Biosciences, San Jose, CA) per product instructions. Untreated whole blood was processed as a control. LPS + whole blood (100ul) 37 o C, 10min 65ul fix buffer RT, 10min 1ml lysis buffer 1ml wash buffer, spin & aspirate Optional: 1ml 50% methanol, on ice for 10min, spin & aspirate Cell staining with surface & intracellular Abs RT, 30 min, dark Add 0.5ml resuspension buffer and acquire samples with a flow cytometer GM-CSF + whole blood (100ul) 37 o C, 10min 65ul fix buffer RT, 10min 1ml lysis buffer 1ml wash buffer, spin & aspirate Cell staining with surface Abs RT, 30 min, dark 1ml cold ( in NaCl 0.9%) Ice for 10min, spin down & aspirate Cell staining with intracellular Abs RT, 30 min, dark Add 0.5ml resuspension buffer and acquire samples with a flow cytometer Samples were then stained for surface and intracellular markers, followed by resuspension in a stabilizing buffer. Antibody conjugates were all from Beckman Coulter: anti-cd14-pc7 (clone 116), anti-cd45-krome Orange (clone J.33), anti-phospho ERK-Alexa Fluor 488 (clone E10, T202/Y204), anti-phospho Akt-Alexa Fluor 647 (clone D9E, S473), anti-phospho P38-Alexa Fluor 647 (clone 28B10, T180/Y182), anti-phospho S6-Pacific Blue (clone D57.2.2E, S235/236), anti-phospho STAT5-Alexa Fluor 647 (clone C71E5, Y694). Data from these samples were collected on a Gallios Cytometer and analyzed with Kaluza software (Beckman Coulter). The signal to noise ratio (S/N) was calculated as (MFI of activated cells) / (MFI of control cells). 3

5 RESULTS perk-af488 pp38-af647 pakt-af488 ps6-af647 S/N 5.02 S/N S/N 2.24 S/N No MeOH, with wash S/N 5.40 S/N S/N 1.84 S/N Donor 1 With MeOH % of CD14+ cells S/N 6.98 S/N S/N 2.53 S/N No MeOH, with wash S/N 6.94 S/N S/N 2.19 S/N Donor 2 With MeOH Figure 1. The staining signals of pp38, perk, pakt or ps6 are similar for samples regardless of 50% methanol treatment when using the PerFix-p reagent. Whole blood from six healthy donors were activated with LPS as described; control samples were not activated. The samples were processed either with or without 50% methanol treatment. Samples were stained with CD14- PC7 and CD45-Krome Orange, and with either perk + pp38 or pakt + ps6. Monocytes were gated using CD45 and CD14. The histograms show overlays of activated (red) and control (brown) samples from 2 donors, and are labeled with S/N values. pp38-af647 pakt-af647 perk-af488 ps6-pacific Blue % of CD14+ cells S/N S/N 6.30 S/N S/N Figure 2. PerFix-p kit enables the multicolor detection of pp38, pakt, perk, and ps6 without methanol treatment in LPS activated monocytes. 4

6 Whole blood samples from two healthy donors were activated with LPS; control samples were not activated. The samples were processed with PerFix-p buffers without methanol treatment. Samples were stained with CD14-PC7 and CD45-Krome Orange, and either with perk-af488 + pakt-af647 + ps6-pacific Blue or with pp38-af647. Monocytes were gated using CD45 and CD14. Example histograms show overlays of activated (red) and control (brown) samples from, and are labeled with S/N values. Donor 2 Side Scatter Phosflow CD14-PC7 PerFix-p kit PerFix-p kit S/N values FIX&PERM kit Donor pp perk PerFix-p kit Phosflow kit Donor pp perk Figure 3. Performance of PerFix-p reagents versus reagents from other vendors. Whole blood from two healthy donors were activated with LPS; control samples were not activated. Samples were processed with PerFix-p reagents as described (without methanol). Donor 1 was also processed with FIX&PERM reagents and donor 2 was also processed with Phosflow reagents; both kits used as per product instructions. Samples were stained with CD14-PC7 and with either pp38-af488 or perk-af488. The histograms show example CD14 staining with either PerFix-p or Phosflow reagents. The table shows S/N values achieved with the two donors. Control Overlay Side Scatter Count S/N 17 + GM-CSF CD45-Krome Orange CD14-PC7 pstat5-af647 Figure 4. PerFix-p reagents enable the intracellular detection of pstat5 in GM-CSF-activated monocytes. Whole blood of a healthy donor was activated with GM-CSF; control samples were not activated. The samples were stained with CD14-PC7 and CD45-Krome Orange before treatment, and were stained with pstat5-af647 following treatment. Monocytes were gated on CD45 and CD14. The histogram overlay shows activated (red) and control (brown) populations. 5

7 No-GM-CSF With-GM-CSF Overlay No wash before MeOH SS INT % of CD14+ cells With wash before MeOH CD14 pstat5-af647 Figure 5. The requirement for a wash step between lysis and treatment. Whole blood from a healthy donor was activated with GM-CSF; control samples were not activated. Samples were processed either with or without adding a wash step after cell lysis step and before treatment. Samples were then stained with CD14-PC7, CD45-Krome Orange, and pstat5-af647. The overlays of activated (red) and control (brown) populatons were gated on monocytes (CD14 + ). No methanol Before After MFI % MFI % MFI % No GM-CSF Side Scatter MFI % MFI % MFI % With GM-CSF CD14-PC7 Figure 6. Detection of CD14 is improved by staining before treatment. Whole blood from a healthy donor was activated with or without GM-CSF as described. A wash step was added after cell lysis step for all samples. The samples was stained with CD14 without methanol treatment, before 80% methanol treatment, or after treatment. The pstat5 stainings (not shown) are all performed after the treatment. The CD14% and MFI of CD14 are indicated on CD14/SS plots. 6

8 Donor 1 Donor 2 Donor 3 PBMC PBMC PBMC pstat5 AF647 - LPS pstat5 Staining Count Whole blood Whole blood Whole blood S/N S/N Donor 1 Donor 2 Donor 3 Donor 1 Donor 2 Donor 3 PBMC WBC pstat5-af647 Figure 7. The staining signals of pstat5 are similar for whole blood or peripheral blood monocyte cells (PBMC) samples when use PerFix-p reagents. Whole blood or blood-derived PBMC (Ficoll separation) from three healthy donors were activated with GM-CSF; control samples were not activated. The samples were stained with CD14-PC7 and CD45-Krome Orange before methanol-treatment step, and then stained with pstat5-af647 after treatment. The overlays show monocytes from activated (red) and control (brown) samples for the 3 donors. The bar graph shows the pstat5 S/N ratios for PBMC (blue) and whole blood (red) for each donor. in PBS (from 1x PBS) in PBS (from 10X PBS) in 0.9% NaCl S/N 10.3 S/N 13.0 S/N 12.8 % of CD14 + cells S/N 13.9 S/N 16.3 S/N 17.2 Donor 1 Donor 2 pstat5-af647 Figure 8. Methanol (80%) for pstat5 detection can perform better when prepared in saline versus PBS. Whole blood samples (triplicate tubes) from two normal donors were activated with GM-CSF; control samples were not activated. Samples were stained with CD14-PC7 and CD45-Krome Orange before methanol treatment and with pstat5-af647 after treatment. The s was prepared with PBS from 1X or 10X stocks, or with 0.9% NaCl. Data overlays show of activated (red) and control (brown) monocyte populations; histograms are labeled with S/N values. In addition, methanol prepared in NaCl (0.9%) was less likely to form precipitates when stored at -20 o C. 7

9 SUMMARY PerFix-p reagents enable the methanol-free detection of some intracellular phospho-proteins (pp38, perk, ps6, pakt) using flow cytometry. PerFix-p reagents display superior signal-to-noise in detecting surface (CD14) & phospho-proteins ( pp38 & perk) than products from other vendors PerFix-p kit enables detection of pstat5 protein in monocytes activated with GM-CSF when used with 80% methanol. Surface markers that are damaged by methanol treatment (such as CD14) may be detected by staining with conjugate prior to the treatment. Similar pstat5 staining signals (S/N) are detected when either whole blood or same donor blood-derived PBMC is activated with GM-CSF and then processed with PerFix-p reagents. Methanol (80%) prepared in NaCl (0.9%) buffer is more stable and enables an at least equivalent or superior pstat5 signal detection than a methanol (80%) prepared in PBS buffer. PerFix-p kit can be shipped or stored in an ambient conditions (room temperature) and with a shelf life at least one year. REFERENCES Chow S, Hedley D, Grom P, Magarri R, Jacobberger J and Shankey V. A whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of phospho-epitope expression in leukocyte subpopulations. Cytometry 67A: 4-17, Chow S, Minden MD, and Hedley DW. Constitutive phosphorylation of the S6 ribosomal protein via mtor and ERK signalling in the peripheral blasts of acute leukemia patients. Exptl. Haematol. 34: , 2006 Woost PG, Solchaga LA, Meyerson HJ, Shankey TV, Goolsby CL, Jacobberger JW. High-resolution kinetics of cytokine signaling in human CD34/CD117- positive cells in unfractionated bone marrow. Blood 117: 131-e141, Marvin J, Swaminathan S, Kraker G, Chadburn A, Jacobberger J, Goolsby C. Normal bone marrow signal-transduction profiles: a requisite for enhanced detection of signaling dysregulations in AML. Blood 117:e120-e130, 2011 ACKNOWLEDGMENTS We would like to thank the extended team that supported the development of the PerFix-p kit: Karen Fischer, Bruce Chin, Yetty Ivan, Fabrice Malergue, Sylvain Monseaux, Laeticia Khemici, Maxime Thiebaut, Nelly Lachenmaier, David Bossy, Corine Manetti, Brigitte Komaïgorodsky, Christian Bovier Lapierre, Isabelle Martin, Christine Athenour, Daniel Desbiolles, Mohamed Rekik, Christophe Rodeville & Jean Cyril Gouin. Gallios, Kaluza, and Krome Orange are trademarks of Beckman Coulter, Inc. Beckman Coulter, VersaLyse and the stylized logo are registered trademarks of Beckman Coulter, Inc and are registered in the USPTO. Pacific Blue is a trademark of Molecular Probes, Inc. Alexa Fluor is a registered trademark of Molecular Probes, Inc. *PerFix-p, Gallios and Kaluza are for Research Use Only. Not for use in diagnostic procedures. 8

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