Experiment Overview
| Repository ID: | FR-FCM-ZYEB | Experiment name: | An optimized multiplex flow cytometry protocol for the analysis of intracellular signaling in peripheral blood mononuclear cells | MIFlowCyt score: | 83.75% |
| Primary researcher: | Richard Davies | PI/manager: | Richard Davies | Uploaded by: | Richard Davies |
| Experiment dates: | 2015-01-21 - 2015-02-20 | Dataset uploaded: | Nov 2017 | Last updated: | Apr 2018 |
| Keywords: | [flow cytometry] [PBMC] [signaling] [Barcoding] [Multiparameter] [Phosphorylation] | Manuscripts: | [27369043] | ||
| Organizations: |
University of Bergen, Bergen, (Norway)
University of Bergen, Clinical Institute 2, Bergen, (Norway) |
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| Purpose: | To evaluate the robustness of a 3 panel FC barcoded phosphoflow panel for the measurement of ERK1/2, NFkB p65, p38, Stat1 S727, Stat1 Y701, Stat3 S727, Stat3 Y705 in monocytes, T cells, NK cells, CD3+CD56+ cells and B cell. Analysis was conducted using cryopreserved PBMC from 3 donors, with 3 repeat measures conducted over a 1 month period. | ||||
| Conclusion: | The data indicated excellent precision of measurements (as assessed by the coefficient of variation, calculated against the 3 replicant). The robustness of the assay was shown to vary based on stimulus, cell type and phospho-protein measured; but in most instances were found to be lower than 10%. | ||||
| Comments: | 9x barcoding stimulus combinations are as follows- POlow + PB low= Unstimulated, POlow + PBmid= IFNalpha (100ng/ml), POlow + PBhigh= IL-2 (100ng/ml), POmid + PBlow= IL-6 (100ng/ml), POmid + PBmid= PMA (100ng/ml), POmid + PBhigh = IL-10 (100ng/ml), POhigh + PBlow = IFNgamma (100ng/ml), POhigh + PBmid = LPS (10micrograms/ml), and POhigh + PBhigh= Unstimulated. | ||||
| Funding: | This work was supported by grants from the Bergen Research Foundation, the Broegelmann Foundation and the Meltzer Foundation | ||||
| Quality control: | Samples acquired on a LSRI Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA). CS & T beads were used daily to assess consistency of machine measurements. | ||||
Experiment variables
| Individuals | |
|---|---|
| · C1 | 21012015_C1 BC_011.fcs · 21012015_C1 P1_001_013.fcs · 21012015_C1 P1_005.fcs · 21012015_C1 P2_001_014.fcs · 21012015_C1 P2_007.fcs · 21012015_C1 P3_001_015.fcs · 21012015_C1 P3_009.fcs · 15022015_C1 BC_011.fcs · 15022015_C1 P1_005.fcs · 15022015_C1 P2_007.fcs · 15022015_C1 P3_009.fcs · 20022015_C1 BC_011.fcs · 20022015_C1 P1_005.fcs · 20022015_C1 P2_007.fcs · 20022015_C1 P3_009.fcs |
| · C2 | 21012015_C2 BC_004.fcs · 21012015_C2 P1_001.fcs · 21012015_C2 P2_002.fcs · 21012015_C2 P3_003.fcs · 15022015_C2 BC_004.fcs · 15022015_C2 P1_001.fcs · 15022015_C2 P2_002.fcs · 15022015_C2 P3_003.fcs · 20022015_C2 BC_004.fcs · 20022015_C2 P1_001.fcs · 20022015_C2 P2_002.fcs · 20022015_C2 P3_003.fcs |
| · C3 | 21012015_C3 BC_012.fcs · 21012015_C3 P1_006.fcs · 21012015_C3 P2_008.fcs · 21012015_C3 P3_010.fcs · 15022015_C3 BC_012.fcs · 15022015_C3 P1_006.fcs · 15022015_C3 P2_008.fcs · 15022015_C3 P3_010.fcs · 20022015_C3 BC_012.fcs · 20022015_C3 P1_006.fcs · 20022015_C3 P2_008.fcs · 20022015_C3 P3_010.fcs |
