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Katharina Kronenberg

Paloma Riquelme

James A. Hutchinson

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Standard protocols for immune profiling of peripheral blood leucocyte subsets by flow cytometry using DuraClone IM reagents

This document describes standard protocols used by the Hutchinson group for analysis of human blood leucocyte populations by flow cytometry (1). DuraClone reagents are dry pre-formulated antibody panels for extensive phenotyping of T cells, B cells, NK cells, dendritic cells and granulocytes in whole blood. In our experience, DuraClone IM Tubes are a convenient, reliable and highly standardised option for immune monitoring of clinical trials. In part, these protocols were adapted from the publications of Streitz, Kverenland and colleagues (2-3), as well as manufacturer’s recommendations issued by Beckman Coulter.

ReagentsDuraClone IM Phenotyping Basic Tube (B53309, Beckman Coulter)DuraClone IM T cell subsets Tube (B53328, Beckman Coulter)DuraClone IM TCRs Tube (B53340, Beckman Coulter)DuraClone IM Granulocytes Tube (B88651, Beckman Coulter)DuraClone IM Dendritic Cells Tube (B53351, Beckman Coulter)DuraClone IM B cells Tube (B53318, Beckman Coulter)DuraClone IM Treg Tube (B53346, Beckman Coulter)VersaLyse Solution (A09777, Beckman Coulter)DPBS without Ca2+ or Mg2+ (D8537, Sigma)IOTest 3 Fixative Solution (10x) (A07800, Beckman Coulter)FBS South American (10270106, Thermo Fisher), heat-inactivatedEcotainer Aqua B, Distilled water (0082423E, Braun)PerFix-nc Kit (B31168, Beckman Coulter)&nbsp;MaterialsS-Monovette® 2,7 ml, K3 EDTA (05.1167, Sarstedt)15 ml tube, PP, 17/120 mm, conical bottom (188271, Greiner Bio-One)

EquipmentNavios cytometer: Standard configuration with 10 colours, 3 lasers (Beckman Coulter)Cytometry List Mode Data Acquisition and Analysis Software (Beckman Coulter)Kaluza analysis software v.1.3 (Beckman Coulter)

1. Preparation of reagents1.1 Preparation of 0.8% IOTest 3 Fixative Solution 1.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Dilute 100 µl IOTest 3 Fixative Solution (10X) with 900 µl PBS to obtain&nbsp;0.8 % IOTest 3 Fixative Solution &nbsp;1.2 Preparation of 0.1% IOTest 3 Fixative Solution1.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Dilute 12.5 µl IOTest 3 Fixative Solution (10X) with 987.5 µl PBS to obtain 0.1 % IOTest 3 Fixative Solution &nbsp;1.3 Preparation of 1X PerFix-nc Buffer 3 1.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Prepare a 1/10 dilution of 10X Buffer 3 in ddH20: dilute 1 part 10X Buffer 3 with 9 parts of ddH2O&nbsp;2. Sample preparation using DuraClone IM Tubes:&nbsp;Phenotyping Basic, T cell subsets, TCRs and Dendritic Cells1.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Collect peripheral blood samples directly into EDTA-vacutainers2.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Samples can be stored at 4 °C for a maximum of 4 h before processing3.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Mix the blood sample by repeatedly inverting the vacutainer4.4.1.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;For DuraClone IM Phenotyping Basic, T cell, TCRs and Granulocytes Tube: transfer 100 µl blood into the DuraClone Tube4.2.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;For DuraClone IM Dendritic Cells Tube: transfer 200 µl blood into the DuraClone Tube5.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Vortex tube for 8 seconds6.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Incubate at room temperature in the dark for 15 min7.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Add 2 ml VersaLyse Solution8.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Vortex tube for 3 seconds9.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Incubate at room temperature in the dark for 15 min10.&nbsp;Pellet cells by centrifugation at 200 g for 5 min11.&nbsp;Aspirate supernatant using a vacuum pump12.&nbsp;Vortex tube for 3 seconds13.&nbsp;Add 3 ml DPBS14.&nbsp;Pellet cells by centrifugation at 200 g for 5 min15.&nbsp;Aspirate supernatant using a vacuum pump16.&nbsp;Resuspend cell pellet in 380 µl 0.8 % IOTest 3 Fixative Solution for data collection using a Navios flow cytometer 3. Sample preparation using DuraClone IM Tubes: B cells1.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Collect peripheral blood samples directly into EDTA-vacutainers2.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Samples can be stored at 4 °C for a maximum of 4 h before processing3.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Mix the blood sample by repeatedly inverting the vacutainer4.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Transfer 10 ml DPBS into a 15 ml conical bottom tube5.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Add 300 µl blood to the 10 ml DPBS6.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Mix by inverting the tube7.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Pellet cells by centrifugation at 300 g for 10 min8.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Aspirate supernatant using a vacuum pump9.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Vortex tube for 3 seconds10.&nbsp;Add 10 ml DPBS11.&nbsp;Mix by inverting the tube12.&nbsp;Pellet cells by centrifugation at 300 g for 5 min13.&nbsp;Aspirate supernatant using a vacuum pump14.&nbsp;Resuspend cell pellet in DPBS to get a final volume of 300 µl15.&nbsp;Transfer 100 µl of washed blood sample into the DuraClone IM B cells Tube16.&nbsp;Vortex tube for 8 seconds17.&nbsp;Incubate at room temperature in the dark for 15 min18.&nbsp;Add 2 ml VersaLyse19.&nbsp;Vortex for 3 seconds20.&nbsp;Incubate at room temperature in the dark for 15 min21.&nbsp;Pellet cells by centrifugation at 200 g for 5 min22.&nbsp;Aspirate supernatant using a vacuum pump23.&nbsp;Vortex tube for 3 seconds24.&nbsp;Add 3 ml DPBS25.&nbsp;Pellet cells by centrifugation at 200 g for 5 min26.&nbsp;Aspirate supernatant using vacuum a pump27.&nbsp;Resuspend cells in 380 µl 0.1 % IOTest 3 Fixative Solution for data collection using a Navios flow cytometer 4. Sample preparation using DuraClone IM Tubes: Tregs1.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Collect peripheral blood samples directly into EDTA-vacutainers2.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Samples can be stored at 4 °C for a maximum of 4 h before processing 3.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Mix the blood sample by repeatedly inverting the vacutainer 4.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Transfer 50 µl blood into the DuraClone IM Treg Tube 15.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Vortex for 8 seconds6.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Incubate at room temperature in the dark for 15 min7.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Add 3 ml DPBS8.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Pellet cells by centrifugation at 500 g for 5 min9.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Aspirate supernatant using vacuum pump10.&nbsp;Vortex for 3 seconds11.&nbsp;Add 50 µl heat-inactivated FCS 12.&nbsp;Mix by pipetting13.&nbsp;Add 5 µl PerFix-nc Buffer 114.&nbsp;Vortex for 8 seconds15.&nbsp;Incubate at room temperature in the dark for 15 min16.&nbsp;Add 400 µl PerFix-nc Buffer 217.&nbsp;Vortex for 8 seconds18.&nbsp;Transfer content of DuraClone IM Treg Tube 1 into DuraClone IM Treg Tube 2 by pipetting19.&nbsp;Vortex Tube 2 for 8 seconds20.&nbsp;Incubate at room temperature in the dark for 60 min21.&nbsp;Add 3 ml DPBS22.&nbsp;Incubate at room temperature in the dark for 5 min23.&nbsp;Pellet cells by centrifugation at 500 g for 5 min24.&nbsp;Aspirate supernatant using a vacuum pump25.&nbsp;Vortex for 3 seconds26.&nbsp;Add 3 ml 1X PerFix-nc Buffer 3 27.&nbsp;Pellet cells by centrifugation at 500 g for 5 min28.&nbsp;Aspirate supernatant using a vacuum pump29.&nbsp;Resuspend cells in 380 µl 1X PerFix-nc Buffer 3 for data collection using a Navios flow cytometer Analysis of data obtained using DuraClone IM TubesTemplate manual gating strategies for extracting leucocyte subset frequencies from flow cytometry data using Kaluza software are given in Figure 1 to 7. Analyses are ideally performed by experienced operators blinded to clinical outcomes.Fig. 1 DuraClone IM Phenotyping Basic Tube Fig. 2 DuraClone IM T cell subsets Tube Fig. 3 DuraClone IM TCRs Tube Fig. 4 DuraClone IM Granulocytes Cells Tube Fig. 5 DuraClone IM &nbsp;Dendritic Cells Tube Fig. 6 DuraClone IM B cells Tube Fig. 7 DuraClone IM Treg Tube

1. Hutchinson, JA. Predicting Early Viral Control under Direct-Acting Antiviral Therapy for Chronic Hepatitis C Virus Using Pretreatment Immunological Markers. Front Immunol. 7;9:146 (2018) 2. Kverneland, AH. Age and gender leucocytes variances and references values generated using the standardized ONE-Study protocol. Cytometry A. 89(6):543-64 (2016)3. Streitz, M. et al. Standardization of whole blood immune phenotype monitoring for clinical trials: panels and methods from the ONE study. Transplant Res. 25;2(1):17 (2013)

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This document describes standard protocols used by the Hutchinson group for profiling human peripheral blood leucocytes by flow cytometry analyses using DuraClone IM reagents.

Standard protocols for immune profiling of peripheral blood leucocyte subsets by flow cytometry using DuraClone IM reagents

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Method Article

Katharina Kronenberg, Paloma Riquelme, James A. Hutchinson

Katharina Kronenberg

Department of Surgery, University Hospital Regensburg, Germany

Paloma Riquelme

Department of Surgery, University Hospital Regensburg, Germany

Corresponding Author

ORCiD: https://orcid.org/0000-0001-8962-3156

James A. Hutchinson

Department of Surgery, University Hospital Regensburg, Germany

ORCiD: https://orcid.org/0000-0002-1199-4210

DOI:

10.21203/rs.3.pex-757/v1

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Flow Cytometry, Immune monitoring, DuraClone, IM Phenotyping Basic Tube, IM T cell subsets Tube, IM TCRs Tube, IM Granulocytes Tube, IM Dendritic Cells Tube, IM B cells Tube, IM Treg Tube

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Method Article

Katharina Kronenberg, Paloma Riquelme, James A. Hutchinson

Katharina Kronenberg

Department of Surgery, University Hospital Regensburg, Germany

Paloma Riquelme

Department of Surgery, University Hospital Regensburg, Germany

Corresponding Author

ORCiD: https://orcid.org/0000-0001-8962-3156

James A. Hutchinson

Department of Surgery, University Hospital Regensburg, Germany

ORCiD: https://orcid.org/0000-0002-1199-4210

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10.21203/rs.3.pex-757/v1

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