The phosphorylation of p38MAPK, Akt and ERK was measured as the Median Fluorescence Intensity (MFI) and normalized using Cytocalbeads (Thermo Scientific). MFI values of the unstimulated samples were subtracted from the stimulated MFI values. Data and statistical analysis was performed with Diva-version 6.0 software (BD Biosciences) and Graph Pad Prism 5.0 (Graph Pad Software Inc., La Jolla, CA) by using paired t-tests (for the in vitro phosphorylation study after performing log transformation and after finding a p-value >0.05 with an F-test). A two-sided p-value < 0.05 was considered statistically significant.
Cytocalbeads
Manufactured by Thermo Fisher Scientific
Sourced in United States
Cytocalbeads are uniform, fluorescently-labeled polystyrene beads designed for cell counting, sizing, and analysis applications. The beads are available in a range of sizes and fluorescent dye options to suit different experimental requirements.
Automatically generated - may contain errors
7 protocols using cytocalbeads
1
Phosphorylation of MAPK Signaling Pathway
2
Quantitative Fluorescent Reporter Assay
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HEK293T cells were double transfected with splice donor (N-GFP) and splice acceptor P1, P2, N1, N2, Lib, or Scr (C-GFP). In 24-well format, 500 ng DNA and 1.0 µL Lipofectamine were used for transfection, which was performed in triplicate. Seventy-two hours post-transfection, cells were analyzed in a flow cytometer (BD Accuri C6). The MESF ratio of GFP was normalized to iRFP (splice donor) and mRFP (splice acceptor). CytoCal beads (Thermo Fisher Scientific) were used for MESF normalization.
3
Quantitative phosphorylation analysis
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The Median Fluorescence Intensity (MFI) was measured for the phosphorylation of p38MAPK, ERK and Akt and data-analysis was performed with Diva-version 6.0 software (BD Bioscience). Negative values measured with flow cytometry can be explained by the compensation settings of the FACS and are displayed via hyperlog transformation49 (link). MFI values were normalized using Cytocalbeads (Thermo Scientific). Statistical analysis was performed with Graph Pad Prism 5.0 (Graph Pad Software Inc., La Jolla, CA) by using paired and unpaired t-tests (after finding a p-value > 0.05 with the Kolmogorov-Smirnov test for normality for the study population). Correlations between drug concentrations and phosphorylation were calculated as the Pearson correlation coefficient. Associations between phosphorylation levels and co-variates were tested by linear regression with IBM SPSS statistics software (version 21; IBM Analytics, Chicago, Illinois, USA). Bonferroni correction was used to correct for multiple testing. A two-sided p-value < 0.05 was considered statistically significant and for the association calculations, two-sided p-values of <0.0042 were considered statistically significant after Bonferroni correction.
4
Quantifying NFATc1 Expression in Cells
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The median fluorescence intensity (MFI) of NFATc1 was measured and data-analysis was performed with Diva-version 6.0 software (BD Bioscience). MFI values were normalized using Cytocalbeads (Thermo Scientific). To calculate the total amplification of the inducible NFATc1/A isoform, samples were further analyzed by correcting the stimulated total expression of NFATc1 (both phosphorylated and dephosphorylated) for the MFI value in unstimulated samples (also both phosphorylated and dephosphorylated). Statistical analysis was performed with Graph Pad Prism 5.0 (Graph Pad Software Inc., La Jolla, CA) by using paired and unpaired t-tests (after finding a p-value > 0.05 with the Kolmogorov-Smirnov test for normality of the study population). Correlations between drug concentrations and the expression of NFATc1 were calculated as the Spearman correlation coefficient. A two-sided p-value < 0.05 was considered statistically significant.
5
Optimizing Flow Cytometry Instrument Calibration
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Machine QC was performed according to the protocol developed by Perfetto et al. 3 . Briefly, optimal voltage settings of PMTs were determined in two steps: first, by defining the separation of dimly stained Cytocal beads (Thermo Fisher Scientific, Waltham, MA, USA) over unstained compensation beads (Compbeads; BD Biosciences, San Jose, CA, USA) at 50 V intervals (range 350–800 V), and second, by defining the separation of quantum simply cellular beads (QSCB; previously stained with fluorescently conjugated monoclonal antibodies) over unstained Compbeads at ±25 V of the target voltage defined in the first step. Inter‐experiment QC was performed by running single‐peak Rainbow beads (Spherotec, Lake Forest, IL, USA) and unstained Compbeads and by adjusting PMT voltages to reach target values. Laser delays were adjusted manually.
6
Phosphorylation Dynamics of Cell Signaling Pathways
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The Median Fluorescence Intensity (MFI) was measured for the phosphorylation of p38MAPK, ERK, and Akt and data analysis was performed with Diva-version 6.0 software (BD Bioscience). MFI values were normalized using Cytocalbeads (Thermo Scientific). Statistical analysis was performed with Graph Pad Prism 5.0 (Graph Pad Software Inc., La Jolla, CA) by using paired and unpaired t-tests (after finding a p-value > 0.05 with the Kolmogorov-Smirnov test for normality for the study population). Correlations between drug concentrations and phosphorylation were calculated as the Spearman correlation coefficient. Associations between
7
Intracellular Phosphoprotein Analysis by Flow Cytometry
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Copyright Ó 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Steinheim, Germany) was added for 15 minutes to activate the blood cells. Applied final concentrations of PMA/ionomycin were 500 ng per mL/5 µg per mL for samples stained for p38MAPK and Akt, and 100 ng per mL/1 µg per mL was used for ERK, based on prior titration for optimal detection of phosphorylated protein. Thereafter, cells were fixed for 10 minutes with Lyse/Fix buffer (BD Biosciences). After permeabilization with 90% methanol at -20ºC for 30 minutes, intracellular staining was performed with phycoerythrin (PE)-labeled mouse anti-p-p38MAPK (clone pT180/pY182), PE-labeled mouse anti-p-Akt (clone pS473), or AlexaFluor647 (AF647)-labeled mouse anti-p-ERK1/2 (pT202/pY204) mAB (all from BD Biosciences) for 30 minutes at room temperature. Samples were analyzed on a FACS Canto II flow cytometer (BD Biosciences). Isotype controls; mouse IgG1-PE (p38MAPK and Akt, Biolegend) and mouse IgG1-AF647 (ERK; Biolegend); were included in separate tubes and served as negative controls. Interday-variability of the flow cytometer was corrected by using Cytocalbeads (Thermo Scientific, Fremont, CA) according to the manufacturer's instructions.
Steinheim, Germany) was added for 15 minutes to activate the blood cells. Applied final concentrations of PMA/ionomycin were 500 ng per mL/5 µg per mL for samples stained for p38MAPK and Akt, and 100 ng per mL/1 µg per mL was used for ERK, based on prior titration for optimal detection of phosphorylated protein. Thereafter, cells were fixed for 10 minutes with Lyse/Fix buffer (BD Biosciences). After permeabilization with 90% methanol at -20ºC for 30 minutes, intracellular staining was performed with phycoerythrin (PE)-labeled mouse anti-p-p38MAPK (clone pT180/pY182), PE-labeled mouse anti-p-Akt (clone pS473), or AlexaFluor647 (AF647)-labeled mouse anti-p-ERK1/2 (pT202/pY204) mAB (all from BD Biosciences) for 30 minutes at room temperature. Samples were analyzed on a FACS Canto II flow cytometer (BD Biosciences). Isotype controls; mouse IgG1-PE (p38MAPK and Akt, Biolegend) and mouse IgG1-AF647 (ERK; Biolegend); were included in separate tubes and served as negative controls. Interday-variability of the flow cytometer was corrected by using Cytocalbeads (Thermo Scientific, Fremont, CA) according to the manufacturer's instructions.
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