Experiment Overview
| Repository ID: | FR-FCM-Z2NR | Experiment name: | Stabilization of human whole blood samples for multi-center and retrospective immunophenotyping studies - Mass cytometry, staining Panel B | MIFlowCyt score: | 71.75% |
| Primary researcher: | Paulina Rybakowska | PI/manager: | Concepcion Maranon | Uploaded by: | Paulina Rybakowska |
| Experiment dates: | 2019-01-31 - 2020-03-06 | Dataset uploaded: | Jun 2020 | Last updated: | Oct 2020 |
| Keywords: | [flow cytometry] [mass cytometry] [immune monitoring] [Whole blood preservation] | Manuscripts: | [33070416] [33070416] |
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| Organizations: |
GENYO. Centre for Genomics and Oncological Research: Pfizer / University of Granada / Andalusian Regional Government, Granada, Choose a State or Province (Spain)
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| Purpose: | This .fcs files form the part of the manuscript titled “Stabilization of human whole blood samples for multi-center and retrospective immunophenotyping studies”. The .fcs files presented in this experiment were obtained for mass cytometry (MC) part of the manuscript, using antibody Panel B. To verify long time of storage, 45 aliquots of whole blood from a single donor using PROT protocol were fixed and frozen, and single aliquots were stained regularly using a 34-plex panel that expands both surface and cytokine detection | ||||
| Conclusion: | The use of a reference sample in MC studies becomes necessary when multiple barcoded batches are run. This reference sample can then be used with confidence to normalize data across multiple runs. Our study provides the way to preserve a reference sample up to 13 months, showing remarkable sample stability in both phenotyping and cytokine studies. | ||||
| Comments: | The reference blood sample was stimulated in a big volume with 1.25 μg/ml of R848 in the presence of Protein Transport Inhibitor Cocktail. 45 aliquots were fixed and frozen using PROT protocol, and single aliquots were stained regularly using a 34-plex panel that expands both surface and cytokine detection (Table S2, panel B in the manuscript). These files contain normalized, cleaned and live leukocytes events. Detailed gating strategy can be found in figure S9 in the manuscript. | ||||
| Funding: | Not disclosed | ||||
| Quality control: | Cells were acquired on a mass cytometer (HELIOS, Fluidigm) at an event rate of 300-350 cells/second together with EQ Four Element Calibration Beads (Fluidigm). Raw data were normalized using normCytof function from CATALYST package. After noticing some signal instability in the data, we decided to run two selected functions from the flowAI package (flow_rate_bin, flow_rate_check) to remove regions with unstable flowrate. To make this function applicable to MC data, we adapted the TIMESTEP to 1 bin per 10 seconds. flowCut algorithm was used to remove signal instability with 1000 segments and MaxPercCut to 0.5. FlowJo software v10.0 was used to analyze normalized MC data by manual gating. Normalized, cleaned and live leukocytes events were selected using Pt and exported to this .fcs files. | ||||
Experiment variables
| Timepoints | |
|---|---|
| · EXPERIMENT 1 | CYT1_REF.fcs |
| · EXPERIMENT 2 | CYT2_REF.fcs |
| · EXPERIMENT 3 | CYT3_REF.fcs |
| · EXPERIMENT 4 | CYT4_REF.fcs |
| · EXPERIMENT 5 | CYT5_REF.fcs |
| · EXPERIMENT 6 | CYT6_REF.fcs |
| · EXPERIMENT 7 | CYT7_REF.fcs |
| · EXPERIMENT 8 | CYT8_REF.fcs |
| · EXPERIMENT 9 | CYT9_REF.fcs |
| · EXPERIMENT 10 | CYT10_REF.fcs |
| · EXPERIMENT 11 | CYT11_REF.fcs |
| · EXPERIMENT 12 | CYT12_REF.fcs |
| · EXPERIMENT 13 | CYT13_REF.fcs |
| · EXPERIMENT 14 | CYT14_REF.fcs |
| · EXPERIMENT 15 | CYT15_REF.fcs |
| · EXPERIMENT 16 | CYT16_REF.fcs |
| · EXPERIMENT 17 | CYT17_REF.fcs |
| · EXPERIMENT 18 | CYT18_REF.fcs |
| · EXPERIMENT 19 | CYT19_REF.fcs |
| · EXPERIMENT 20 | CYT20_REF.fcs |
| · EXPERIMENT 21 | CYT21_REF.fcs |
| · EXPERIMENT 22 | CYT22_REF.fcs |
| · EXPERIMENT 23 | CYT23_REF.fcs |
| · EXPERIMENT 24 | CYT24_REF.fcs |
| · EXPERIMENT 25 | CYT25_REF.fcs |
| · EXPERIMENT 26 | CYT26_REF.fcs |
| · EXPERIMENT 27 | CYT27_REF.fcs |
| · EXPERIMENT 28 | CYT28_REF.fcs |
| · EXPERIMENT 29 | CYT29_REF.fcs |
| · EXPERIMENT 30 | CYT30_REF.fcs |
| · EXPERIMENT 31 | CYT31_REF.fcs |
| · EXPERIMENT 32 | CYT32_REF.fcs |
| · EXPERIMENT 33 | CYT33_REF.fcs |
| · EXPERIMENT 34 | CYT34_REF.fcs |
| · EXPERIMENT 35 | CYT35_REF.fcs |
| · EXPERIMENT 36 | CYT36_REF.fcs |
| · EXPERIMENT 37 | CYT37_REF.fcs |
| · EXPERIMENT 38 | CYT38_REF.fcs |
| · EXPERIMENT 39 | CYT39_REF.fcs |
| · EXPERIMENT 40 | CYT40_REF.fcs |
| · EXPERIMENT 41 | CYT41_REF.fcs |
| · EXPERIMENT 42 | CYT42_REF.fcs |
| · EXPERIMENT 43 | CYT43_REF.fcs |
| · EXPERIMENT 44 | CYT44_REF.fcs |
| · EXPERIMENT 45 | CYT45_REF.fcs |
