Tumor Immune Cells Isolation and Analysis

Tumors were harvested, mechanically cut into small pieces, and then enzymatically treated with 1 mg/mL collagenase D (IV type, Clostridium histolyticum, Sigma) and 0.02 mg/mL DNase I (from bovine pancreas grade 2, Roche) at 37 °C for 30 min. The enzymatic reaction was stopped by adding EDTA (Sigma). The ensuing single-cell suspension was filtered by means of a 70-µm cell strainer and prepared for cytofluorimetric analysis. After Fc blocking, single-cell suspensions were stained with the following antibodies from Miltenyi Biotec: CD45-VioGreen (clone: REA737); NK1.1-APC (clone: PK136); CD11b-Vioblue (clone: REA592); CD3-PE (clone: REA641); CD69-PEVio770 (clone: REA937); Ly6C-PE (clone: 1G7.G10); CD4-PEVio770 (clone: REA604); CD8-Vioblue (clone: 53.6.7); and F4/80-PercPVio700 (clone: REA126). Subsequently, cell viability was determined by LIVE/Dead-633 nm (L10120) staining according to the manufacturer’s instructions (Invitrogen); negative cells were considered viable. Doublet exclusion and gating on live CD45⁺ cells were performed. The following subpopulations of CD45⁺ were identified: CD11b⁺ Ly6C⁺ (monocytes), CD11b⁺ F4/80⁺ (macrophages), CD3⁺CD4⁺ and CD3⁺C8⁺ (T cells) and NK1.1⁺CD3⁻ (NK cells). Cells were analyzed on a MACSQuant16 (Miltenyi) and analyzed with FlowJo software (RRID:SCR_008520, Treestar) [28 (link)].

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Girone C., Calati F., Lo Cigno I., Salvi V., Tassinari V., Schioppa T., Borgogna C., Lospinoso Severini L., Hiscott J., Cerboni C., Soriani A., Bosisio D, & Gariglio M. (2023). The RIG-I agonist M8 triggers cell death and natural killer cell activation in human papillomavirus-associated cancer and potentiates cisplatin cytotoxicity. Cancer Immunology, Immunotherapy, 72(9), 3097-3110.

Publication 2023

collagenase D DNase I EDTA CD45-VioGreen NK1.1-APC CD11b-Vioblue CD3-PE Ly6C-PE CD4-PEVio770 CD8-Vioblue F4/80-PercPVio700 MACSQuant16

Antibodies Bovine Cd11b Cell viability Cells Clone Clostridium histolyticum Collagenase Dnase Edta Enzymatic Macrophages Monocytes Nk cells Pancreas Subpopulations T cells Tumors

Corresponding Organization :

Other organizations : Università degli Studi del Piemonte Orientale “Amedeo Avogadro”, University of Brescia, Sapienza University of Rome, Istituto Pasteur

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Variable analysis

independent variables

Mechanical cutting of tumors into small pieces
Enzymatic treatment with 1 mg/mL collagenase D and 0.02 mg/mL DNase I at 37 °C for 30 min

dependent variables

Subpopulations of CD45+ cells: CD11b+ Ly6C+ (monocytes), CD11b+ F4/80+ (macrophages), CD3+CD4+ and CD3+CD8+ (T cells), and NK1.1+CD3− (NK cells)

control variables

Fc blocking
LIVE/Dead-633 nm staining for cell viability
Doublet exclusion
Gating on live CD45+ cells

positive controls

Not mentioned

negative controls

Not mentioned

Annotations

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