Experiment Overview
| Repository ID: | FR-FCM-Z68G | Experiment name: | A Flow Cytometry Panel to Investigate Human Neutrophil Subsets in Health and Disease | MIFlowCyt score: | 68.28% |
| Primary researcher: | Craig Schofield | PI/manager: | Luke Garratt | Uploaded by: | Craig Schofield |
| Experiment dates: | 2023-02-22 - 2023-03-15 | Dataset uploaded: | Apr 2023 | Last updated: | Jul 2023 |
| Keywords: | [Neutrophil] [human] [migration] [subset] [ontogeny] [granule release] [immune modulation] | Manuscripts: | [38179854] |
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| Organizations: |
Wal Yan Respiratory Research Centre, Telethon Kids Institute, Nedlands, Western Australia (Australia)
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| Purpose: | This 14-colour, 13-antibody OMIP was designed for deep profiling of neutrophil subsets in various types of human samples to contextualise neutrophil plasticity in a range of healthy and diseased states. Markers present in the OMIP allow the profiling of neutrophil subsets associated with ontogeny, migration, phagocytosis capacity, granule release, and immune modulation. For panel design, we ensured that the commonly available fluorophores FITC/AF488 and PE were assigned to the intracellular subset marker Olfactomedin 4 and maturity marker CD10 respectively. These markers can be easily replaced without affecting core identification of neutrophils, enabling antibodies to new neutrophil antigens of interest or for fluorescent substrates to assess different neutrophil functions to be easily explored. Panel optimisation was performed on whole blood and purified neutrophils. We demonstrate applications on clinical samples (whole blood) and experimental endpoints (purified neutrophils stimulated through an in vitro transmigration assay). We hope that providing a uniform platform to analyse neutrophil plasticity in various sample types will facilitate the future understanding of neutrophil subsets in health and disease. | ||||
| Conclusion: | The flexible application of this panel combined with the capability for widespread adaptation to different human samples will drive much needed contextualisation of neutrophil subsets in human health and disease. | ||||
| Comments: | FCS files are for three experiments: FMOs, Migration experiment and saliva experiment. Each was acquired on the following dates: FMOs - 22/02/2023 Migration experiment - 02/03/2023 Saliva experiment - 15/03/2023 There are three copies of the compensation beads. Also uploaded is the Compensation matrices for each experiment. There are three copies of the calibration beads labeled with the experiment date. The beads were acquired and matched to target values, then recorded as CB0. CB1-3 were acquired throughout the course of the experiment to monitor instrument drift. | ||||
| Funding: | This research was funded by the National Health and Medical Research Council (NHMRC) Project Grant (Award# 1142505), Telethon Kids Institute Plus 10 award, as well as the Western Australian Future Health Research and Innovation Fund, which is an initiative of the WA State Government. LWG is a NHMRC Early Career Fellow (Award# 1141479). | ||||
| Quality control: | Ultra Rainbow Calibration Kit, 6 peaks URCP-38-2K Spherotech Whole Blood FMOs Ultracomp E beads compensation beads | ||||
Experiment variables
| Conditions | |
|---|---|
| · Fluorescence Minus One experiment | 230222_OMIP_5thGen_CD10_PE_FMO_016.fcs · 230222_OMIP_5thGen_CD11b_BUV737_FMO_010.fcs · 230222_OMIP_5thGen_CD15_BV786_FMO_005.fcs · 230222_OMIP_5thGen_CD16_BUV395_FMO_011.fcs · 230222_OMIP_5thGen_CD177_APC_FMO_009.fcs · 230222_OMIP_5thGen_CD184_BV421_FMO_015.fcs · 230222_OMIP_5thGen_CD45_R718_FMO_006.fcs · 230222_OMIP_5thGen_CD49d_BV480_FMO_013.fcs · 230222_OMIP_5thGen_CD54_BUV469_FMO_007.fcs · 230222_OMIP_5thGen_CD62L_PE-Cy7_FMO_008.fcs · 230222_OMIP_5thGen_CD63_BV650_FMO_014.fcs · 230222_OMIP_5thGen_CD66b_PE-Fire660_FMO_012.fcs · 230222_OMIP_5thGen_FVS780_FMO_017.fcs · 230222_OMIP_5thGen_FullStain_Stim_019.fcs · 230222_OMIP_5thGen_FullStain_Unstim_020.fcs · 230222_OMIP_5thGen_OLFM-4_AF488_FMO_018.fcs · 230222_OMIP_5thGen_Unstained_021.fcs |
| · Migration experiment | 230302_OMIP_5thGen_PS195_Psa_Mig_006.fcs · 230302_OMIP_5thGen_PS195_Uninfected_Mig_005.fcs · 230302_OMIP_5thGen_PS195_WB_FullStain_013.fcs · 230302_OMIP_5thGen_PS196_Psa_Mig_008.fcs · 230302_OMIP_5thGen_PS196_Uninfected_Mig_007.fcs · 230302_OMIP_5thGen_PS196_WB_FullStain_014.fcs · 230302_OMIP_5thGen_PS197_PSA_Mig_010.fcs · 230302_OMIP_5thGen_PS197_Uninfected_Mig_009.fcs · 230302_OMIP_5thGen_PS197_WB_FullStain_015.fcs |
| · 230222_FMO_CalibrationBeads | Voltage_Set_Ultra Rainbow CB0_001_230222.fcs · Voltage_Set_Ultra Rainbow CB1_002_230222.fcs · Voltage_Set_Ultra Rainbow CB2_003_230222.fcs · Voltage_Set_Ultra Rainbow CB3_004_230222.fcs |
| · 230302_Migration_CalibrationBeads | Voltage_Set_Ultra Rainbow CB0_001_230302.fcs · Voltage_Set_Ultra Rainbow CB1_002_230302.fcs · Voltage_Set_Ultra Rainbow CB2_003_230302.fcs · Voltage_Set_Ultra Rainbow CB3_004_230302.fcs |
| · 230315_Saliva_CalibrationBeads | Voltage_Set_Ultra Rainbow CB0_230315_001.fcs · Voltage_Set_Ultra Rainbow CB1_230315_002.fcs · Voltage_Set_Ultra Rainbow CB2_230315_003.fcs |
| Sample Type | |
|---|---|
| · Stimulated Whole Blood | 230222_OMIP_5thGen_CD10_PE_FMO_016.fcs · 230222_OMIP_5thGen_CD11b_BUV737_FMO_010.fcs · 230222_OMIP_5thGen_CD15_BV786_FMO_005.fcs · 230222_OMIP_5thGen_CD16_BUV395_FMO_011.fcs · 230222_OMIP_5thGen_CD177_APC_FMO_009.fcs · 230222_OMIP_5thGen_CD184_BV421_FMO_015.fcs · 230222_OMIP_5thGen_CD45_R718_FMO_006.fcs · 230222_OMIP_5thGen_CD49d_BV480_FMO_013.fcs · 230222_OMIP_5thGen_CD54_BUV469_FMO_007.fcs · 230222_OMIP_5thGen_CD62L_PE-Cy7_FMO_008.fcs · 230222_OMIP_5thGen_CD63_BV650_FMO_014.fcs · 230222_OMIP_5thGen_CD66b_PE-Fire660_FMO_012.fcs · 230222_OMIP_5thGen_FVS780_FMO_017.fcs · 230222_OMIP_5thGen_FullStain_Stim_019.fcs · 230222_OMIP_5thGen_OLFM-4_AF488_FMO_018.fcs · 230222_OMIP_5thGen_Unstained_021.fcs |
| · Naive Whole Blood | 230222_OMIP_5thGen_FullStain_Unstim_020.fcs · 230302_OMIP_5thGen_PS195_WB_FullStain_013.fcs · 230302_OMIP_5thGen_PS196_WB_FullStain_014.fcs · 230302_OMIP_5thGen_PS197_WB_FullStain_015.fcs |
| · Transmigrated Neutrophils | 230302_OMIP_5thGen_PS195_Psa_Mig_006.fcs · 230302_OMIP_5thGen_PS195_Uninfected_Mig_005.fcs · 230302_OMIP_5thGen_PS196_Psa_Mig_008.fcs · 230302_OMIP_5thGen_PS196_Uninfected_Mig_007.fcs · 230302_OMIP_5thGen_PS197_PSA_Mig_010.fcs · 230302_OMIP_5thGen_PS197_Uninfected_Mig_009.fcs · Voltage_Set_Ultra Rainbow CB3_004_230222.fcs |
| · Calibration Beads | Voltage_Set_Ultra Rainbow CB0_001_230222.fcs · Voltage_Set_Ultra Rainbow CB0_001_230302.fcs · Voltage_Set_Ultra Rainbow CB0_230315_001.fcs · Voltage_Set_Ultra Rainbow CB1_002_230222.fcs · Voltage_Set_Ultra Rainbow CB1_002_230302.fcs · Voltage_Set_Ultra Rainbow CB1_230315_002.fcs · Voltage_Set_Ultra Rainbow CB2_003_230222.fcs · Voltage_Set_Ultra Rainbow CB2_003_230302.fcs · Voltage_Set_Ultra Rainbow CB2_230315_003.fcs · Voltage_Set_Ultra Rainbow CB3_004_230302.fcs |
| Individuals | |
|---|---|
| · Migration Experiment Donor 1 | 230302_OMIP_5thGen_PS195_Psa_Mig_006.fcs · 230302_OMIP_5thGen_PS195_Uninfected_Mig_005.fcs · 230302_OMIP_5thGen_PS195_WB_FullStain_013.fcs |
| · Migration Experiment Donor 2 | 230302_OMIP_5thGen_PS196_Psa_Mig_008.fcs · 230302_OMIP_5thGen_PS196_Uninfected_Mig_007.fcs · 230302_OMIP_5thGen_PS196_WB_FullStain_014.fcs |
| · Migration Experiment Donor 3 | 230302_OMIP_5thGen_PS197_PSA_Mig_010.fcs · 230302_OMIP_5thGen_PS197_Uninfected_Mig_009.fcs · 230302_OMIP_5thGen_PS197_WB_FullStain_015.fcs |
