Comprehensive Immune Profiling by Flow Cytometry

Flow cytometry analysis was performed as previously described (20 (link)). Single cells were stained with Alexa Fluor 594 dye (Thermo Fisher), to exclude dead cells from analysis, and then fixed in paraformaldehyde (PFA; 1.6%). Fixed cells were stained with the following antibodies: CD3-Pacific Blue (clone UCHT1), CCR7-PE (150503), CXCR5-Ax488 (RF8B2), CD20-APCH7 (L27), PDL1-APC (MIH1), PDL2-APC (MIH18), and IFNγ-PE (4S.B3) from BD Biosciences; TIGIT-APC (MBSA43), LAG3-PeCy7 (3DS223H), TNFα-Ax488 (MAb11), and IL2-PeCy7 (MQ1–17H12) from eBioscience; and CD4-Ax700 (RPA-T4), CD8-Bv785 (RPA-T8), CD45RA-Bv510 (HI100), PD1-Bv650 (EH12.2H7), TIM3-APC (F38–2E2), BTLA-APC (MIH26), CD244-PerCPCy5.5 (C1.7), CD160-PeCy7 (BY55), LAIR1-PerCPCy5.5 (NKTA255), CD155-PE (SKII.4), and CD112-PeCy7 (TX31) from BioLegend. Brilliant Stain Buffer (BD Biosciences) was used as staining buffer. Data were acquired on LSR II (BD Biosciences) and analyzed using Cytobank (https://www.cytobank.org/).

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Josefsson S.E., Beiske K., Blaker Y.N., Førsund M.S., Holte H., Østenstad B., Kimby E., Köksal H., Wälchli S., Bai B., Smeland E.B., Levy R., Kolstad A., Huse K, & Myklebust J.H. (2019). TIGIT and PD-1 Mark Intratumoral T Cells with Reduced Effector Function in B-cell Non-Hodgkin Lymphoma. Cancer immunology research, 7(3), 355-362.

Publication 2019

Alexa Fluor 594 dye IFNγ-PE (4S.B3) ), TIM3-APC ( Brilliant Stain Buffer

Alexa 594 Antibodies Btla Buffer Cd112 Cd155 Cells Clone Cxcr5 Flow cytometry Ifnγ Paraformaldehyde Pdl1 Stain Tigit Tim3 Tnfα

Corresponding Organization : University of Oslo

Other organizations : Karolinska Institutet

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Variable analysis

independent variables

Staining with Alexa Fluor 594 dye to exclude dead cells
Staining with the following antibodies: CD3-Pacific Blue, CCR7-PE, CXCR5-Ax488, CD20-APCH7, PDL1-APC, PDL2-APC, IFNγ-PE, TIGIT-APC, LAG3-PeCy7, TNFα-Ax488, IL2-PeCy7, CD4-Ax700, CD8-Bv785, CD45RA-Bv510, PD1-Bv650, TIM3-APC, BTLA-APC, CD244-PerCPCy5.5, CD160-PeCy7, LAIR1-PerCPCy5.5, CD155-PE, CD112-PeCy7

dependent variables

Expression levels of the cell surface markers and intracellular cytokines measured by flow cytometry

control variables

Paraformaldehyde (PFA; 1.6%) used to fix the cells
Brilliant Stain Buffer (BD Biosciences) used as staining buffer

controls

Positive control: Not explicitly mentioned
Negative control: Excluding dead cells from analysis by staining with Alexa Fluor 594 dye

Annotations

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